Xavier Vidal-Gómez

Aging-related endothelial dysfunction in the aorta from female senescence-accelerated mice is associated with decreased nitric oxide synthase expression

The present study investigated the time-course for aging-associated effects on contractile and relaxing vascular responses and nitric oxide (NO) production in the aorta from female senescence-accelerated resistant (SAMR1) and prone (SAMP8) mice. Both SAMR1 and SAMP8 were studied at three different ages: 3 (young), 6 (middle age) and 10 (old) months. Concentration-response curves to phenylephrine (10(-8) to 10(-5) M) or acetylcholine (10(-9) to 10(-5) M) were performed in the aortic rings in the absence or in the presence of NO synthase (NOS) inhibitor L-NAME (10(-4) M). Protein and gene expression for endothelial NOS (eNOS) was determined by immunofluorescence, Western blot and real-time PCR. Although we have not seen any difference in vascular responses when comparing both strains at 3 months old, we found a significant aging-associated impairment of vascular reactivity that follows a distinct time-course in SAMR1 and SAMP8. In SAMR1, increases in phenylephrine contraction and decreases in acetylcholine relaxation were only seen at 10 months old, while SAMP8 displays altered responses at 6 months that are further impaired at 10 months old. L-NAME treatment enhanced phenylephrine contractions and completely inhibited acetylcholine relaxations in all age groups of SAMR1 and SAMP8. However, the magnitude of increase in phenylephrine contraction by L-NAME was markedly reduced by aging and followed a faster pace in SAMP8. Similar pattern of responses was observed in the time course for changes of eNOS expression, suggesting an earlier and more pronounced aging-associated decrease of NO production and eNOS expression in SAMP8. These results reveal that aging enhances contractile responses to phenylephrine and decreases endothelium-dependent relaxation to acetylcholine in the aorta from female mice by a mechanism that involves a decrease of NO production. This process occurs earlier in the aorta from SAMP8 mice, establishing these mice as suitable model to study cardiovascular aging in a convenient and standard time course.

Estradiol, acting through ERα, induces endothelial non-classic renin-angiotensin system increasing angiotensin 1-7 production.

Intracellular renin-angiotensin system (RAS) can operate independently of the circulating RAS. Estrogens provide protective effects by modulating the RAS. Our aim was to investigate the effect of estradiol (E2) on angiotensin converting enzymes (ACE) 1 and ACE2 expression and activities in human endothelial cells (HUVEC), and the role of estrogen receptors (ER). The results confirmed the presence of active intracellular RAS in HUVEC. Physiological concentrations of E2 induced a concentration-dependent increase of ACE1 and ACE2 mRNA expression and ACE1, but not ACE2, protein levels. ACE1 and ACE2 enzymatic activities were also induced with E2. These effects were mediated through ERα activation, since ER antagonists ICI 182780 and MPP completely abolished the effect of E2. Moreover, the ERα agonist PPT mirrored the E2 effects on ACE1 and ACE2 protein expression and activity. Exposure of endothelial cells to E2 significantly increased Ang-(1-7) production. In conclusion, E2 increases Ang-(1-7) production, through ERα, involving increased ACE1 and ACE2 mRNA expression and activity and ACE1 protein levels.

Decreased bioavailability of nitric oxide in aorta from ovariectomized senescent mice. Role of cyclooxygenase

This study investigates the effects of aging and/or ovariectomy on vascular reactivity to thromboxane A2 (TXA2) receptor stimulation with U46619, and the modulation by nitric oxide (NO) and cyclooxygenase (COX) in aorta from female senescence-accelerated mice (SAMP8) and from senescence resistant mice (SAMR1). Five-month-old female SAMR1 and SAMP8 were divided into three groups: sham-operated, ovariectomized and ovariectomized plus estradiol. Twenty-eight days after surgery, thoracic aortic rings were mounted for isometric recording of tension and concentration-response curves for U46619 (10(-10)-3 × 10(-7) M) were performed in the absence and in the presence of the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 10(-4) M) and/or COX inhibitor indomethacin (10(-5)M). Vascular superoxide production was detected by dihydroethidium staining on sections of thoracic aorta. NO bioavailability in response to U46619 was suppressed by estrogen withdrawn in young and senescent mice and was restored by the administration of estradiol. In the presence of indomethacin, contractions to U46619 decreased in all groups indicating an aging- and estrogen-dependent modulation of contractile prostanoids. The simultaneous incubation of L-NAME and indomethacin did not change the maximal responses and sensitivities to TXA2 in any group in comparison with untreated aortic segments. The superoxide generation induced by TXA2 was greater in aorta from SAMP8 than in SAMR1. Moreover, in ovariectomized groups superoxide production was further increased and treatment with 17β-estradiol reverted the effects of the ovariectomy. Inhibition of COX with indomethacin prevented the U46619-induced increase in superoxide formation. Our results indicate that NO bioavailability in response to TP receptor activation is both estrogen- and aging-dependent. TXA2 induced contractions are partially mediated by COX activation. Both aging and ovariectomy enhanced COX-dependent component of the TXA2-induced contraction. It is noteworthy that in the absence of estrogen, COX inhibition induces an increase of NO bioavailability. Therefore, in senescent female mice with an experimental menopause, TP-receptor stimulation is responsible for COX activation and enhanced superoxide generation, which may result in reduced NO bioavailability. These effects were reversed by estrogen administration.

miRNA as a New Regulatory Mechanism of Estrogen Vascular Action

The beneficial effects of estrogen on the cardiovascular system have been reported extensively. In fact, the incidence of cardiovascular diseases in women is lower than in age-matched men during their fertile stage of life, a benefit that disappears after menopause. These sex-related differences point to sexual hormones, mainly estrogen, as possible cardiovascular protective factors. The regulation of vascular function by estrogen is mainly related to the maintenance of normal endothelial function and is mediated by both direct and indirect gene transcription through the activity of specific estrogen receptors. Some of these mechanisms are known, but many remain to be elucidated. In recent years, microRNAs have been established as non-coding RNAs that regulate the expression of a high percentage of protein-coding genes in mammals and are related to the correct function of human physiology. Moreover, within the cardiovascular system, miRNAs have been related to physiological and pathological conditions. In this review, we address what is known about the role of estrogen-regulated miRNAs and their emerging involvement in vascular biology.

MicroRNA as Crucial Regulators of Gene Expression in Estradiol-Treated Human Endothelial Cells

Background/Aims: Estrogen signalling plays an important role in vascular biology as it modulates vasoactive and metabolic pathways in endothelial cells. Growing evidence has also established microRNA (miRNA) as key regulators of endothelial function. Nonetheless, the role of estrogen regulation on miRNA profile in endothelial cells is poorly understood. In this study, we aimed to determine how estrogen modulates miRNA profile in human endothelial cells and to explore the role of the different estrogen receptors (ERα, ERβ and GPER) in the regulation of miRNA expression by estrogen. Methods: We used miRNA microarrays to determine global miRNA expression in human umbilical vein endothelial cells (HUVEC) exposed to a physiological concentration of estradiol (E2; 1 nmol/L) for 24 hours. miRNA-gene interactions were computationally predicted using Ingenuity Pathway Analysis and changes in miRNA levels were validated by qRT-PCR. Role of ER in the E2-induced miRNA was additionally confirmed by using specific ER agonists and antagonists. Results: miRNA array revealed that expression of 114 miRNA were significantly modified after E2 exposition. Further biological pathway analysis revealed cell death and survival, lipid metabolism, reproductive system function, as the top functions regulated by E2. We validated changes in the most significantly increased (miR-30b-5p, miR-487a-5p, miR-4710, miR-501-3p) and decreased (miR-378h and miR-1244) miRNA and the role of ER in these E2-induced miRNA was determined. Results showed that both classical, ERα and ERβ, and membrane-bound ER, GPER, differentially regulated specific miRNA. In silico analysis of validated miRNA promoters identified specific ER binding sites. Conclusion: Our findings identify differentially expressed miRNA pathways linked to E2 in human endothelial cells through ER, and provide new insights by which estrogen can modulate endothelial function.

2C.04: TIMING OF BLOOD PRESSURE AND VASCULAR CHANGES INDUCED BY AGEING IN AORTA AND SMALL MESENTERIC ARTERIES FROM FEMALE SENESCENCE-ACCELERATED MOUSE PRONE (SAMP8).

Objective: Age is the most important risk factor for cardiovascular diseases. A key requisite to develop new interventions for age-related conditions is the availability of preclinical murine models. We propose prone senescence-accelerated mice (SAMP8) to study vascular ageing in a convenient and standard time course. Our aim was to investigate the effects of ageing on blood pressure and endothelium-dependent relaxation in large and small arterial vessels of female SAMP8 in order to characterize the vascular changes in this experimental model of ageing. Design and method: Female SAMP8 mice at 3-, 6- and 10-months old (n = 8 in each group) were studied. Thoracic aorta (∼1 mm internal diameter) and small mesenteric arteries (SMA ∼200 &mgr;m internal diameter) were mounted for isometric recording of tension. The endothelium-dependent relaxations to acetylcholine (ACh, 10–9 to 10–5 M) were performed in the absence and in the presence of the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 10–4 M). Blood pressure (n = 6 in each group) was measured using the tail-cuff method. Histological analysis were carried out by hematoxylin-eosin staining. Results: A decrease in endothelium-dependent relaxation to ACh in SAMP8 aorta was observed at 6-months old (91 ± 3 vs 72 ± 4%, p < 0.05) and further decrease was observed at 10-month old (72 ± 4 vs 64 ± 4%, p < 0.05). In contrast, in SMA the relaxation decreased at 10-months old (98 ± 4% vs 89 ± 4%, p < 0.05). In aorta, the ACh-induced relaxation was completely inhibited by L-NAME, but in SMA were reduced a 38 ± 4% suggesting than other endothelial-derived relaxant factors, distinct from NO, could counterbalance the decreased NO biavailability induced by ageing in small arteries. An increment in blood pressure and hemodynamic parameters are observed at 10-months old. Conclusions: Ageing induces earlier endothelial dysfunction in aorta than in SMA suggesting that aortic endothelial cells are more sensitive to deleterious effects of ageing. The enhancement of blood pressure match with a decreased endothelium-dependent relaxation in small arteries. Our results support the use of SAMP8 mice to study ageing-associated vascular function in females.

[PP.14.27] COMPARATIVE ANALYSIS OF MIRNA EXPRESSION IN SERUM AND PLASMA OF PATIENTS WITH ACUTE MYOCARDIAL INFARCTION

Objective: miRNAs are present in human biofluids and have been pointed as potential biomarkers for diagnosis and assessment of prognosis of different cardiovascular diseases. Nevertheless, previous reports have suggested that different methodological variances could affect miRNA concentration. In this study we aim to compare the expression of major miRNAs associated with acute myocardial infarction (AMI) in plasma and serum from Non-ST-elevation myocardial infarction (NSTEMI) patients relative to samples from healthy volunteers. Design and method: Plasma and corresponding serum samples were collected from healthy individuals (n = 20) and NSTEMI patients (n = 40) prior to coronary angioplasty. Blood was collected into anti-coagulant free (for serum) and EDTA Vacutainer® (for plasma) tubes. Circulating miRNA was purificated using miRCURY RNA Isolation Kit (Exiqon). 50 ng of RNA was used for reverse transcription of the selected miRNAs (miRNA-21, miRNA-26a, miRNA-92a, and miRNA-133a). TaqMan MicroRNA Assays were used to assess the expression of individual miRNAs. Relative quantification was performed using QuantStudio Software. Values were normalized to the expression of the reference gene RNU6 and miRNA-484. Results: miRNA yield was similar in samples from plasma and serum. The expression level of RNU6 and miR-484 was similar in samples from plasma and serum. Nevertheless, we observed a higher variability of RNU6 in plasma than in serum, and we chose miRNA-484 as endogenous control. miRNA-26a decreased in samples from AMI patients showing the same pattern expression in serum and plasma (36 ± 5% and 18 ± 6% of control values, respectively, p < 0.01). miRNA-133a increased in serum (482 ± 78%, p < 0.01) and showed a slight increase in plasma (157% of control values, p > 0.05) from AMI patients. miRNA-92a expression was decreased only in plasma samples (33 ± 4%, p < 0.01), and miR-21 showed an opposite pattern expression, being 10-fold increased in serum (p < 0.05) and decreased in plasma (32 ± 9%, p < 0.05). Conclusions: Plasma and serum exhibit different pattern of circulating miRNA expression in patients with AMI and suggest that results from studies with different starting material could not be comparable. These results call our attention to the importance of maintaining methodological care in when determining the expression of miRNA in patients.

[PP.20.02] CHANGES IN MICRO-RNA PROFILE AND MICRO-RNA/MESSENGER-RNA REGULATORY NETWORKS IN HUMAN ENDOTHELIAL CELLS EXPOSED TO ESTRADIOL

Objective: Estradiol (E2) exerts a key role in the modulation of vascular function, mainly through endothelium-mediated actions. MicroRNAs (miRNA) are small non-coding RNA that modulate post-transcriptional expression by translation repression or target mRNA degradation. The objective of this study was to determine the cardiovascular pathways regulated by E2-sensitive miRNA and to identify miRNA-mRNA networks in cultured human umbilical vein endothelial cells (HUVEC) by transcriptome analysis. Design and method: HUVEC were exposed to 1 nM E2 for 24 hours and miRNA were isolated by miRNeasy Mini Kit. miRNA expression was performed with GeneChip miRNA 4.0 Array (Affymetrix). Validation of miRNA expression was determined by qRT-PCR. miRNA-mRNA interactions were reconstructed using previous mRNA microarray data using the same conditions (PLoS One. 2009;4:e824.). miRNA-mRNA pairings and canonical pathway analysis were performed using Ingenuity Pathway Analysis software. Results: 120 miRNAs were differentially expressed in E2-treated cells compared to control, 47 were up-regulated and 73 down-regulated. Fold changes of E2-regulated miRNAs range from -1,76 to 2,02. Most significantly changed miRNA (miR-30b-p, miR-487a-5p, miR-4710, miR-501–3p, miR-378 h and miR-1244) were validated by qRT-PCR. Bioinformatic analysis determined specific miRNA-mRNA interactions: 81 miRNAs with 1599 mRNA targets. 82.9% of those mRNA targets were inversely correlated to their respective miRNA. We further filtered our miRNA-mRNA pairings using experimentally observed or highly predicted target correlations and we obtained 73 miRNA targeting 698 mRNAs: 47 miRNA were up-regulated (with a total of 474 mRNAs targets) and 26 were down-regulated (with a total of 224 mRNA targets). Data analysis revealed significantly changed canonical pathways relevant for endothelial function, including ERK/MAPK signalling, integrin signalling and actin cytoskeleton signalling. Conclusions: This study identifies E2-induced changes in miRNA expression profile of human endothelial cells. Global miRNA-mRNA networks obtained by integrative microarray analysis elucidate the biological processes by which E2 regulates vascular function.

[BP.12.04] ESTRADIOL INDUCES ENDOGLIN EXPRESSION THROUGH ESTROGEN RECEPTORS IN HUMAN ENDOTHELIAL CELLS AND IN AORTA FROM A MOUSE MODEL OF EXPERIMENTAL MENOPAUSE

Objective: Endoglin (CD105) is an accessory receptor of TGF&bgr;, which is predominantly expressed in endothelial cells and is involved in proliferation and in angiogenesis processes. Indeed, mutations in the endoglin gene are responsible for hereditary haemorrhagic telangiectasia, characterized by telangiectases in skin and mucosa and arteriovenous malformations in several organs. Microarrays analysis performed by our group in human umbilical vein endothelial cells (HUVEC) exposed to estradiol demonstrated an up-regulation of TGF&bgr; pathway and endoglin expression. Our objectives were to determine 1) the role of estrogen receptors (ER) in the regulation of endoglin expression by estradiol in HUVEC, and 2) the expression of endoglin in aortic tissue from an experimental murine model of menopause. Design and method: HUVEC were exposed to 0,1–10 nM of 17&bgr;-estradiol for 24 h in the absence or in the presence of or ICI 182780 (1 &mgr;M, unspecific antagonist of ER&agr; and ER&bgr;), and MPP (1 &mgr;M, ER&agr; antagonist). Female senescence-accelerated mouse (SAM) were used as an experimental model of menopause which join ageing and lack of estrogens: 5 months-old SAM prone (SAMP8, n = 9) and SAM resistant (SAMR1, n = 9) were separated in three groups: sham-operated, ovariectomized and ovariectomized chronically-treated with estradiol. 28 days after surgery, mice were sacrificed and aorta was isolated. mRNA expression of endoglin was analyzed by qRT-PCR, and protein expression by immunoblotting. Results: In HUVEC, estradiol (1 nM) increased both endoglin mRNA and protein expression by 129 ± 11% and 133 ± 11%, respectively. This effect was abolished in the presence of ER antagonists ICI 182780 and MPP, suggesting an involvement of ER&agr; in endoglin expression. In aortic tissue from mice, not only the lack of estrogens affected endoglin expression, but also the ageing. Aorta from aged mice showed a decreased expression of endoglin and ovariectomy in this group did not affect this expression. However, in control mice, ovariectomy decreased endoglin expression and was reversed after estradiol treatment. Conclusions: Estradiol increases gene and protein expression of endoglin through ER&agr; in HUVEC. Both estradiol and ageing also regulate endoglin expression in aortic vascular tissue from a mouse model of menopause.

[OP.6D.01] ESTRADIOL INCREASES ENDOTHELIAL ANGIOTENSIN-(1-7) PRODUCTION THROUGH ESTROGEN RECEPTOR ALPHA.

Objective: Over-activity of the classic renin angiotensin system (RAS) has been implicated in the pathophysiology of cardiovascular diseases primarily through the vasoconstrictor and inflammatory properties of Angiotensin (Ang)II. The non-classic RAS may counteract some AngII effects through its diversion to synthesize the vasodilator Ang-(1-7). Wealth of evidence shows that estrogens provide vascular protective effects by modulating the RAS. We previously reported estradiol (E2) effects on endothelial gene expression profile, demonstrating an increase in ACE1. Our aims were to analyze the E2 effect on ACE1 and ACE2 expression and activities in human umbilical vein endothelial cells (HUVEC) and the role of estrogen receptors (ER). Design and method: Primary HUVEC were exposed to physiological concentrations (1–10 nM) of E2 for 24 hours. Cells were also exposed to specific agonists of ERalpha (PPT) and ERbeta (DPN) and to antagonists of ER (unspecific, ICI 182780, and selective for ERalpha, MPP) or to lisinopril (ACE1 inhibitor) to analyze their role on the observed effects. ACE1 and ACE2 expressions were analyzed by RT-PCR and immunoblotting. Their enzymatic activities were measured by fluorometry by using specific substrates. AngII and Ang-(1-7) production was detected by immunofluorescence. Results: HUVEC exposure to E2 resulted in a dose-dependent increase of both ACE1 and ACE2 mRNA expression. This ACE1 increment was confirmed with the amount of protein, while ACE2 protein expression remained unaltered. ACE1 and ACE2 enzymatic activities were also increased with E2. These effects were mediated through ERalpha activation, since ICI 182780 and MPP completely abolished the E2 effect. Exposure of HUVEC to E2 significantly increased Ang-(1-7) production. Treatment with MPP and lisinopril reverted the E2 mediated effects. Conclusions: E2 increases Ang-(1-7) endothelial production by enhancing its local enzymatic regulation through ERalpha. Thus, our results add new information that supports a role for ERalpha in E2-mediated vasodilatory effects. Supported by the Spanish Ministerio de Economía y Competitividad, ISCIII – FEDER-ERDF (grants FIS PI13/00091, PI13/00617, and RD12/0042/0052). D.P-C. is an ‘Atracció de Talent’ fellow (PREDOC13-110541, Univ. Valencia). A.M. is a ‘Formación de Profesorado Universitario’ fellow (FPU13/02235 Spanish Min. Educación, Cultura y Deporte).