Structural, mechanical and myogenic properties of small mesenteric arteries from ApoE KO mice: Characterization and effects of virgin olive oil diets

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ Esta es la versión aceptada del artículo publicado en: This is an accepted manuscript of a paper published in: Atherosclerosis: January (2015)  DOI: https://doi.org/10.1016/j.atherosclerosis.2014.11.022 Copyright: Copyright © 2025 Elsevier Inc., its licensors, and contributors. El acceso a la versión publicada del artículo puede requerir la suscripción de la revista. Access to the published version may require subscription. “This is an Accepted Manuscript of an article published by Elsevier in Atherosclerosis 2015, available at: https://doi.org/10.1016/j.atherosclerosis.2014.11.022 ”. Elsevier Editorial System(tm) for Atherosclerosis Manuscript Draft Manuscript Number: Title: Structural, mechanical and myogenic properties of small mesenteric arteries from ApoE KO mice: characterization and effects of virgin olive oil diets Article Type: Basic Research Section/Category: Basic Research - Vascular Biology, Hemostasis, Oxidative Stress Keywords: arterial wall properties, atherosclerosis, small mesenteric artery, virgin olive oil Corresponding Author: Dr. Rosalia Rodriguez-Rodriguez, Corresponding Author's Institution: School of Pharmacy, University of Seville First Author: Elena Ogalla Order of Authors: Elena Ogalla; Carmen Claro; Maria Alvarez de Sotomayor; Maria D Herrera; Rosalia Rodriguez-Rodriguez Suggested Reviewers: Ulf Simonsen Pharmacology, University of Aarhus [email protected] Wide experience on vascular biology, particularlly with small mesenteric arteries in animal models of cardiovascular diseases such as ApoE ko mice. Kim Dora Pharmacology, University of Oxford [email protected] Long expertise on microvascular physiology (function, mechanics, myogenic response) and pharmacology in animal models of disease like ApoE KO mice. Jesus Osada Pharmacology, University of Zaragoza [email protected] Experience on animal models of atherosclerosis and its vascular characterization, particularly in the effect of the Mediterranean diet and virgin olive oil. Elisardo C Vasquez University of Espirito Santo [email protected] Experience on ApoE KO mice and the effect of diet and strain on atherosclerosis Opposed Reviewers: HIGHLIGHTS  This is the first analysis of arterial wall properties in ApoE KO mice small artery.  SMA remodeling and myogenic response in ApoE significantly differed from WT mice.  Virgin olive oil attenuated SMA wall disorders induced by saturated fat-diet.  Endothelial dysfunction was restored by high polyphenols-enriched virgin olive oil.  These results evidence the role of SMA and dietary intervention in atherosclerosis. Highlights (for review) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 1 Structural, mechanical and myogenic properties of small mesenteric arteries from ApoE KO mice: characterization and effects of virgin olive oil diets 5 Elena Ogalla, Carmen Claro, María Alvarez de Sotomayor, Maria Dolores Herrera, Rosalia Rodriguez-Rodriguez* Department of Pharmacology, School of Pharmacy, University of Seville, Seville, Spain 10 *corresponding author: Dr. Rosalia Rodriguez-Rodriguez Department of Pharmacology, School of Pharmacy, University of Seville Postal address: C/ Profesor Garcia-Gonzalez 2. 41012 Seville, Spain. 15 Phone: +34 954557443; Fax: +34 954556074. e-mail: [email protected] *Abstract, Title Page, Manuscript, References, Legends 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 2 ABSTRACT: Objective: We analyzed the structural, mechanical, myogenic and functional properties of resistance arteries of ApoE KO compared to wild type (WT) mice. We also determined the influence of saturated fat in comparison to virgin olive oil-enriched diets in vascular wall abnormalities. 5 Methods: Male ApoE-/- and WT mice (8-weeks-old) were assigned to the groups: standard chow diet (SD), high fat diet (HFD), virgin olive oil (VOO) and high polyphenol-VOO-enriched diet (Oleaster) (OT) (15% w/w). After 20 weeks, structural, mechanical and myogenic properties of isolated small mesenteric arteries (SMA) were analyzed by pressure myography. For functional studies, vasodilatation to acetylcholine was assessed. Arterial superoxide anion 10 production was measured by ethidium fluorescence. Results: Hypertrophic remodeling and distensibility in ApoE SMA was lower compared to WT mice, suggesting an alteration in the autoregulation mechanisms aimed to compensate disease progression. However, ApoE deficiency resulted in a lower impairment in myogenic tone, in addition to an improved endothelium-dependent hyperpolarizing vasodilatation. Also, we 15 evidenced the beneficial effects of VOO in contrast to a saturated fat-enriched diet on SMA wall disorders. Only the endothelial function improvement induced by olive oil was dependent on polyphenols content. Conclusion: Resistance arteries structure, mechanic, myogenic and functional responses from ApoE significantly differ from WT mice, evidencing the influence of the type of diet on these 20 disorders. These results are particularly useful to determine the contribution of resistance arteries during the atherosclerotic process and to provide novel insights into the Mediterranean dietary pattern to reduce the burden of atherosclerotic disease. Keywords: arterial wall properties, atherosclerosis, small mesenteric artery, virgin olive oil 25 HIGHLIGHTS:  This is the first analysis of arterial wall properties in ApoE KO mice small artery.  SMA remodeling and myogenic response in ApoE significantly differed from WT mice.  Virgin olive oil attenuated SMA wall disorders induced by saturated fat-diet. 30  Endothelial dysfunction was restored by high polyphenols-enriched virgin olive oil.  These results evidence the role of SMA and dietary intervention in atherosclerosis. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 3 1. INTRODUCTION The apolipoprotein E (ApoE)-deficient mouse has proved a useful model of human atherosclerosis that rapidly develops atherosclerotic lesions even when maintained on a normal chow diet [1]. Vascular studies with this animal model of atherosclerosis have been mostly focused on largeand medium-size arteries. Specifically, the two main well-known 5 hallmarks in the macrovasculature of ApoE-deficient mice are: (i) vascular dysfunction, which is generally defined by impaired endothelium-dependent vasodilatation prominent on the plaque-rich regions of the aorta [2-5], and (ii) mechanical and structural alterations of the vascular wall in these vessels [6]. However, despite large evidences for endothelial dysfunction and structural and mechanical disorders in conductance arteries, relatively little is known 10 about these processes within the resistance vasculature in humans and animal models of dyslipidemia and atherosclerosis [7]. It has been demonstrated that alterations of resistance arteries reactivity (i.e. endothelial dysfunction) and abnormal small artery remodelling that leads to structural changes, play a crucial role in the pathogenesis of cardiovascular diseases (i.e. ischemic cerebral events, 15 coronary artery disease and renal insufficiency) and have a marked prognostic impact in cardiovascular risk subjects such as those with hypertension, obesity and diabetes [8-12]. Regarding to the microvasculature of ApoE deficient mice, the characterization of structural, mechanical and myogenic properties of small mesenteric arteries remains unexplored. In fact, microvascular studies of this animal model are limited to the investigation of functional 20 properties of small arteries [5, 7, 13-15] revealing the key role of microvessels in atherosclerosis progression. One of the most interesting results was provided by Beleznai et al. [7], showing that dyslipidemia was not detrimental to endothelial function in small mesenteric arteries from ApoE mice, despite advanced atherosclerotic plaque formation in aorta, thus evidencing the interest of resistance arteries and their compensatory role against large arteries 25 dysfunction and hypertrophy in the development of atherosclerosis. Considering the described above, in this study we analyse for the first time the structural, mechanical and myogenic abnormalities of small mesenteric arteries of the commonly used model of atherosclerosis ApoE KO mice in comparison to wild type mice. Additionally, given the implications of the type of diet on cardiovascular diseases and atherosclerosis progression 30 [5, 16], and considering that dietary-based approaches are gaining attention in the progression of microvascular disorders [17-21], the second goal of the present investigation is to compare the effects of a Western type diet with a Mediterranean dietary pattern, specifically virgin olive oil (VOO)-enriched diets, in functional and structural alterations in small arteries from this animal model. This aim is based on the fact that the beneficial effects of olive oil in humans 35 and animal models of atherosclerosis are limited to conductance arteries [16, 22-25], whereas its effects on the microvasculature (in arterial function, mechanics or remodelling) have not been evaluated. Finally, to go further insight into the effect of olive oil in microvascular disorders in atherosclerosis, the effects of traditionally extracted VOO will be compared to Oleaster, which is a VOO prepared by a new process that increases polyphenols content. 40 2. MATERIALS AND METHODS 2.1. Animals and diets 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 4 Male 8 week-old ApoE deficient mice and age-matched C57BL/6J wild-type mice (WT) (Charles River, Barcelona, Spain) were used in this study. The protocol for animal handling and experimentation agreed with the European Union European Community guidelines for the ethical treatment of animals (UE Directive of 2010; 2010/63/UE) and was approved by the Ethical Committee for Animal Research of the University of Seville (RD 53/2013). 5 Animals were fed a high fat diet (40 % Kcal from fat, TD 88137, Harlan, Barcelona, Spain) for 6 weeks. Then, WT and ApoE-/- mice were randomly assigned to one of the following experimental groups (n=10): (1) High fat diet (HFD), (2) virgin olive oil (VOO)-enriched diet (15 %, w/w) and (3) high polyphenol virgin olive oil-enriched diet (Oleaster®) (OT) (15 %, w/w). The oils were supplied by Oleapure S.L. (Sevilla, Spain). Total polyphenols content in VOO was 120 10 ppm whereas in OT was 503 ppm. Diets were weekly prepared and stored at -20ºC. Also, groups of WT and ApoE-/- mice receiving a standard diet (SD) for 20 weeks were established. Animals had ad libitum access to food and water. Food consumption and body weights were weekly recorded throughout the experimental period. At the end of the treatment, animals were kept during 12 h fasting and were anesthetized with chloral hydrate 12 % 15 intraperitoneally. 2.2. Artery isolation and pressure myography The mesentery was collected and placed in cold modified Krebs-Henseleit solution (KHS) (composition in mmol/L: NaCl 118, KCl 4.75, NaHCO3 25, MgSO4 1.2, CaCl2 1.8, KH2PO4 1.18 and glucose 11). Segments of isolated small mesenteric arteries (SMA) were cannulated at each 20 end with glass micropipettes, which were fixed in a pressure myograph (111P, Danish Myo Technology, Aarhus, Denmark), as described [26]. Intraluminal pressure was raised to 120 mmHg and the artery was unbuckled by adjusting the cannulas. The vessel was then set to 70 mmHg pressure and allowed to equilibrate for 30 min at 37ºC in KHS. Intraluminal pressure was reduced to 10 mmHg, and a pressure-diameter curve (10-120 mmHg) was obtained in 25 gassed-KHS. Internal and external diameters (DiCa, DeCa) were measured for 3 minutes at each intraluminal pressure. The artery was then set to equilibrate for 30 min in gassed calcium-free KHS (0Ca2+: omitting calcium and adding 10 mmol/L EGTA). In these conditions, a second pressure-diameter curve was performed [27]. 2.2.1. Calculation of structural, mechanical and myogenic parameters 30 From internal and external diameters measured in passive conditions (0Ca2+) for a given intravascular pressure, the following structural parameters were calculated: wall thickness = (De0Ca – Di0Ca)/2; cross-sectional area (CSA) = (/4) x [(De0Ca)2 – (Di0Ca)2]; and wall to lumen ratio = (De0Ca – Di0ca)/2Di0Ca. To evaluate mechanical properties of the artery, circumferential wall strain () was calculated 35 as (Di0Ca – D00Ca)/D00Ca , where D00Ca is the internal diameter at very low pressure value, 10 mmHg, and Di0Ca is the observed internal diameter for a given intravascular pressure both measured under passive conditions. Also, circumferential wall stress () was determined as (P x Di0Ca)/2wt, where P is the intraluminal pressure (1 mmHg = 133.4 Nm-2) and wt is wall thickness at each intraluminal pressure in 0Ca2+ medium. 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 5 Arterial stiffness independent of geometry is determined by Young’s elastic modulus (E = stress/strain). Since the stress-strain relationship is nonlinear, it is more appropriate to obtain a tangential or incremental elastic modulus by determining the slope of the stress-strain curve (E = /). Elastic modulus was obtained by fitting stress-strain data from each animal to an exponential curve by using the equation  = orig exp (), where orig is the stress at the 5 original diameter (10 mmHg). Taking derivatives on the above equation, E = . For a given value of , elastic modulus is directly proportional to . An increase in  implies an increase in the elastic modulus, which means an increase in stiffness. Therefore, the  value was used as an index of arterial wall stiffness [27]. Myogenic response percentages at each pressure were determined from 100 x DiCa / Di0Ca, 10 where DiCa and Di0Ca are the internal diameters measured in active (1.8 mmol/L Ca2+ - KHS) and passive conditions (0Ca2+ - KHS), respectively. 2.2.2. Assessment of vascular functionality The presence of functional endothelium was determined by the ability of 10 µmol/L acetylcholine (ACh) to induce relaxation in pressurized arterial segments precontracted with 15 phenylephrine (Phe). All experiments were performed in endothelium-intact arteries. After this test, concentration-response curves to ACh (0.001 – 30 mol/L) were constructed in the absence or presence of the following inhibitors at a maximally active concentration [18]: the NOS inhibitor L-NAME (300 mol/L), the non-selective cyclooxygenase inhibitor indomethacin (10 mol/L), alone or in combination for 30 min before the corresponding concentration-20 response curve. Also, curves were made in the presence of the small-conductance calciumactivated potassium (SKCa) channel blocker apamin (Apa, 100 nmol/L) and the blocker of both SKCa and intermediate-conductance KCa (IKCa) channels charybdotoxin (Ctx, 50 nmol/L). These channels are the main mediators of the endothelial-derived hyperpolarizing factor (EDHF) response. 25 2.3. In situ detection of superoxide anion The oxidative fluorescent dye dihydroethidium (DHE) was used to evaluate production of arterial O2 ●- in situ. DHE is oxidized by O2 ●- to yield the red fluorescent DNA stain ethidium. As previously described [21], 14-µm-thick SMA sections placed on gelatin-coated slides were incubated with 2 µmol/L DHE and preparations were viewed by laser scanning confocal 30 microscope (Zeiss LSM 7 Duo; Zeiss, Oberkochen, Germany; 40x objective). Integrated optical densities were quantified using ImageJ software 1.47v (National Institutes of Health, USA). 2.4. Drugs and solutions All drugs were purchased from Sigma Aldrich (St Louis, MO, USA). Indomethacin was dissolved in DMSO. All other stock solutions were dissolved in distilled water. 35 2.5. Statistical analysis Data represented are means ± S.E.M. of the number of mice. Vasodilatation was expressed as a percentage of the previous tone generated by Phe. Two-way analysis of variance followed by Bonferroni's comparison test was used to compare the results. Differences were considered 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 6 significant when P<0.05. A Prism GraphPad Software 5.0 (San Diego, CA, USA) was used for statistical analysis. 3. RESULTS 3.1. Effect of diets on structural and mechanical properties of SMA from WT and ApoE mice As illustrated in figure 1, while WT-HFD showed an important increase in wall thickness and 5 CSA in comparison to WT-SD, these changes associated to HFD were markedly attenuated in atherosclerotic mice. Structural properties of both WT and ApoE mice fed olive oil-enriched diets were significantly altered in comparison to animals fed a HFD, showing similar structural patterns than those observed in SD-groups (Fig. 1). These structural changes induced by VOOand OT-diets were much more evident in ApoE deficient than in WT mice. 10 To evaluate the implication of either the ApoE deficiency or the type of diet in passive mechanical properties of the vascular wall, stiffness, wall stress and strain were analyzed (Fig. 2). When analyzing these parameters, important differences were appreciated between WT and ApoE mice. On the one hand, WT mice fed HFD showed a moderate decrease in arterial stiffness (increased distensibility) as evidenced by a decreased tendency of the  value (Fig. 15 S.1) and a significant rightward shift of the stress-strain relationship compared to the SD group (Fig. 2A). Also, wall stress tended to be attenuated in arteries from WT-HFD, especially at high values of intraluminal pressure (Fig. 2C). In ApoE,  values were significantly higher compared to WT mice and, in contrast to that observed in control arteries, stiffness value tended to increase by HFD administration in atherosclerotic mice (Fig. S.1). Also, and in disparity to WT 20 mice, no significant changes were appreciated in the stress-strain relationship (Fig. 2B) or wall stress (Fig. 2D) between the experimental ApoE groups. Administration of either VOOor OTenriched diets tended to modify mechanical properties of SMA from WT mice as evidenced in SD, whereas these olive oils did not induce significant mechanical changes in ApoE mice. 3.2. Effect of diets on myogenic response of SMA from WT and ApoE mice 25 In active conditions (1.8 mmol/L Ca2+-KHS) internal diameter enlarged from 10 to 60 mmHg and slightly decreased from 60 to 120 mmHg in all the experimental groups. Whereas no differences were appreciated between lumen diameter curves of WT mice groups (Fig. 3A), the increase in internal diameter with intraluminal pressure rising was more evident in ApoE fed a HFD compared to the ApoE-SD group (Fig. 3B). Administration of olive oils significantly 30 attenuated the lumen diameter profile of the curve in ApoE compared to mice fed a HFD (Fig. 3B). The myogenic response is illustrated in figures 3C and 3D, evidencing the extent of the constrictor tone in active relative to passive conditions. In WT mice fed a HFD, myogenic response was markedly reduced at all the intraluminal pressures tested compared to WT-SD 35 (Fig. 3C), indicating an impairment of constrictor tone. In contrast, a lower impairment in the myogenic response of arteries from ApoE mice induced by HFD was evidenced, and this response was only appreciated at high values of intraluminal pressures (Fig. 3D). Olive oilsenriched diets were only able to restore myogenic tone in WT mice at the highest perfusion 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 13 [15] Cola MS, Gava AL, Meyrelles SS, Vasquez EC. Endothelial dysfunction of resistance vessels in female apolipoprotein E-deficient mice. Lipids Health Dis 2010;9:51. [16] Arbonés-Mainar JM, Navarro MA, Carnicer R, Guillén N, Surra JC, Acín S, Guzmán MA, Sarría AJ, Arnal C, Aguilera MP, Jiménez A, Beltrán G, Uceda M, Osada J. Accelerated atherosclerosis in apolipoprotein E-deficient mice fed Western diets containing palm 5 oil compared with extra virgin olive oils: a role for small, dense high-density lipoproteins. Atherosclerosis 2007;194(2):372-82. [17] Agouni A, Lagrue-Lak-Hal AH, Mostefai HA, Tesse A, Mulder P, Rouet P, Desmoulin F, Heymes C, Martínez MC, Andriantsitohaina R. Red wine polyphenols prevent metabolic and cardiovascular alterations associated with obesity in Zucker fatty rats 10 (Fa/Fa). PLoS One 2009;4(5):e5557. [18] Rodriguez-Rodriguez R, Herrera MD, de Sotomayor MA, Ruiz-Gutierrez V. Effects of pomace olive oil-enriched diets on endothelial function of small mesenteric arteries from spontaneously hypertensive rats. Br J Nutr 2009;102(10):1435-44. [19] Davì G, Santilli F, Patrono C. Nutraceuticals in diabetes and metabolic syndrome. 15 Cardiovasc Ther 2010;28(4):216-26. [20] Widmer RJ, Freund MA, Flammer AJ, Sexton J, Lennon R, Romani A, Mulinacci N, Vinceri FF, Lerman LO, Lerman A. Beneficial effects of polyphenol-rich olive oil in patients with early atherosclerosis. Eur J Nutr 2013;52(3):1223-31. [21] Justo ML, Claro C, Vila E, Herrera MD, Rodriguez-Rodriguez R. Microvascular disorders 20 in obese Zucker rats are restored by a rice bran diet. Nutr Metab Cardiovasc Dis 2014;24(5):524-31. [22] Acín S, Navarro MA, Carnicer R, Arbonés-Mainar JM, Guzmán MA, Arnal C, Beltrán G, Uceda M, Maeda N, Osada J. Dietary cholesterol suppresses the ability of olive oil to delay the development of atherosclerotic lesions in apolipoprotein E knockout mice. 25 Atherosclerosis 2005;182(1):17-28. [23] Acín S, Navarro MA, Perona JS, Arbonés-Mainar JM, Surra JC, Guzmán MA, Carnicer R, Arnal C, Orman I, Segovia JC, Osada J, Ruiz-Gutiérrez V. Olive oil preparation determines the atherosclerotic protection in apolipoprotein E knockout mice. J Nutr Biochem 2007;18(6):418-24. 30 [24] Lou-Bonafonte JM, Arnal C, Navarro MA, Osada J. Efficacy of bioactive compounds from extra virgin olive oil to modulate atherosclerosis development. Mol Nutr Food Res 2012;56(7):1043-57. [25] Urpi-Sarda M, Casas R, Chiva-Blanch G, Romero-Mamani ES, Valderas-Martínez P, Arranz S, Andres-Lacueva C, Llorach R, Medina-Remón A, Lamuela-Raventos RM, 35 Estruch R. Virgin olive oil and nuts as key foods of the Mediterranean diet effects on inflammatory biomakers related to atherosclerosis. Pharmacol Res 2012;65(6):577-83. [26] Rodríguez-Rodríguez R, Yarova P, Winter P, Dora KA. Desensitization of endothelial P2Y1 receptors by PKC-dependent mechanisms in pressurized rat small mesenteric arteries. Br J Pharmacol 2009;158(6):1609-20. 40 [27] Caracuel L, Jiménez-Altayó F, Romo M, Márquez-Martín A, Dantas AP, Vila E. Transient mesenteric ischemia leads to remodeling of rat mesenteric resistance arteries. Front Physiol 2012;2:118. [28] Jiménez-Altayó F, Caracuel L, Pérez-Asensio FJ, Martínez-Revelles S, Messeguer A, Planas AM, Vila E. Participation of oxidative stress on rat middle cerebral artery 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 14 changes induced by focal cerebral ischemia: beneficial effects of 3,4-dihydro-6hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran (CR-6). J Pharmacol Exp Ther 2009;331(2):429-36. [29] Trask AJ, Katz PS, Kelly AP, Galantowicz ML, Cismowski MJ, West TA, Neeb ZP, Berwick ZC, Goodwill AG, Alloosh M, Tune JD, Sturek M, Lucchesi PA. Dynamic microand 5 macrovascular remodeling in coronary circulation of obese Ossabaw pigs with metabolic syndrome. J Appl Physiol (1985) 2012;113(7):1128-40. [30] Intengan HD, Schiffrin EL. Vascular remodeling in hypertension: roles of apoptosis, inflammation, and fibrosis. Hypertension 2001;38(3 Pt 2):581-7. [31] Briones AM, Xavier FE, Arribas SM, González MC, Rossoni LV, Alonso MJ, Salaices M. 10 Alterations in structure and mechanics of resistance arteries from ouabain-induced hypertensive rats. Am J Physiol Heart Circ Physiol 2006;291(1):H193-201. [32] Baumbach GL, Didion SP, Faraci FM. Hypertrophy of cerebral arterioles in mice deficient in expression of the gene for CuZn superoxide dismutase. Stroke 2006;37(7):1850-5. 15 [33] McSherry IN, Sandow SL, Campbell WB, Falck JR, Hill MA, Dora KA. A role for heterocellular coupling and EETs in dilation of rat cremaster arteries. Microcirculation 2006;13: 119-130. [34] Cipolla MJ, Gokina NI, Osol G. Pressure-induced actin polymerization in vascular smooth muscle as a mechanism underlying myogenic behavior. FASEB J 2002;16(1):72-20 6. [35] Flavahan NA, Bailey SR, Flavahan WA, Mitra S, Flavahan S. Imaging remodeling of the actin cytoskeleton in vascular smooth muscle cells after mechanosensitive arteriolar constriction. Am J Physiol Heart Circ Physiol 2005;288(2):H660-9. [36] Maneen MJ, Hannah R, Vitullo L, DeLance N, Cipolla MJ. Peroxynitrite diminishes 25 myogenic activity and is associated with decreased vascular smooth muscle F-actin in rat posterior cerebral arteries. Stroke 2006;37(3):894-9. [37] Buil-Cosiales P, Irimia P, Berrade N, Garcia-Arellano A, Riverol M, Murie-Fernández M, Martínez-Vila E, Martínez-González MA, Serrano-Martínez M. Carotid intima-media thickness is inversely associated with olive oil consumption. Atherosclerosis 2008; 30 196(2):742-8. [38] Murie-Fernandez M, Irimia P, Toledo E, Martínez-Vila E, Buil-Cosiales P, SerranoMartínez M, Ruiz-Gutiérrez V, Ros E, Estruch R, Martínez-González MÁ; PREDIMED Investigators. Carotid intima-media thickness changes with Mediterranean diet: a randomized trial (PREDIMED-Navarra). Atherosclerosis 2011;219(1):158-62. 35 [39] Carluccio MA, Massaro M, Scoditti E, De Caterina R. Vasculoprotective potential of olive oil components. Mol Nutr Food Res 2007;51(10):1225-34. STATEMENT OF ORIGINALITY We wish to draw the attention of the Editor to the following facts which may be considered as potential conflicts of interest and to significant financial contributions to this work. We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing we confirm that we have followed the regulations of our institutions concerning intellectual property. We further confirm that any aspect of the work covered in this manuscript that has involved experimental animals has been conducted with the ethical approval of all relevant bodies and that such approvals are acknowledged within the manuscript. We understand that the Corresponding Author is the sole contact for the Editorial process (including Editorial Manager and direct communications with the office). She is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs. We confirm that we have provided a current, correct email address which is accessible by the Corresponding Author and which has been configured to accept email from Rosalia Rodriguez-Rodriguez ([email protected]) Signed by all authors as follows: *Statement of Originality Dear Editor, Enclosed, please find the manuscript entitled “Structural, mechanical and myogenic properties of small mesenteric arteries from ApoE KO mice: characterization and effects of virgin olive oil diets” by Elena Ogalla, Carmen Claro, María Álvarez de Sotomayor, María Dolores Herrera and Rosalia Rodriguez-Rodriguez, which we would like you to consider for publication in Atherosclerosis. I hereby declare that all of the authors listed have contributed to the work and have read, participated in the writing and agreed with the submitted version of the manuscript. The document is original and it has not been published or submitted elsewhere. In addition, the animal studies performed throughout the investigation have been reviewed by the appropriate Ethics Committees. Looking forward to hearing from you. Yours sincerely, Dr. Rosalia Rodriguez-Rodriguez On behalf of all authors Department of Pharmacology, School of Pharmacy, University of Seville. C/ Profesor García González 2. 41012 Seville, Spain. Tel: +34954557443; Fax: +34954556074 e-mail: [email protected] *Submission Letter Suggested Reviewers SUGGESTED REVIEWERS: Prof. Ulf Simonsen Department of Biomedicine - Pharmacology Aarhus University Aarhus, Denmark [email protected] Dr. Kim Dora Department of Pharmacology University of Oxford Mansfield Road, Oxford OX1 3QT, UK Phone: +44 01865 281114 Fax: +44 01865 271853 [email protected] Prof. Jesus Osada Department of Biochemistry and Molecular Biology Veterinary School, University of Zaragoza Miguel Servet 177, E-50013, Zaragoza, Spain Phone: +34 976761644 Fax: +34 976767612 Email: [email protected] Prof. Elisardo C Vasquez Department of Physiological Sciences, Health Sciences Center, Federal University of Espirito Santo, Vitoria, ES, Brazil Correspondence: [email protected] AUTHORS DECLARATION – CONFLICT OF INTEREST We the undersigned declare that this manuscript is original, has not been published before and is not currently being considered for publication elsewhere. We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. We understand that the Corresponding Author is the sole contact for the Editorial process. He/she is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs. Signed by all authors as follows: *Conflict of interest form 020 40 60 80 100 120 10 20 30 40 *** ### ### *** *** WT SD WT HFD WT VOO WT OT Intraluminal pressure (mmHg) Wall thickness (m) 020 40 60 80 100 120 10 20 30 40 *** ApoE OT ApoE VOO ApoE HFD ApoE SD Intraluminal pressure (mmHg) Wall thickness (m) 020 40 60 80 100 120 10000 15000 20000 25000 30000 35000 *** ### ### *** Intraluminal pressure (mmHg) CSA (m2) 020 40 60 80 100 120 10000 15000 20000 25000 30000 35000 ### ### *** Intraluminal pressure (mmHg) CSA (m2) Figure 1 (A) (C) (B) (D) Figure(s) 0.0 0.2 0.4 0.6 0.8 1.0 0 500000 1000000 1.5 1006 2.0 1006 2.5 1006 ApoE HFD ApoE VOO ApoE OT ApoE SD Strain (Di/Do) Stress (dynes cm-2) 0.0 0.2 0.4 0.6 0.8 1.0 0 500000 1000000 1.5 1006 2.0 1006 2.5 1006 WT SD WT HFD WT VOO WT OT Strain (Di/Do) Stress (dynes cm-2) 020 40 60 80 100 120 0 500000 1000000 1.5 1006 2.0 1006 2.5 1006 * Intraluminal pressure (mmHg) Stress (dynes cm-2) *# 020 40 60 80 100 120 0 500000 1000000 1.5 1006 2.0 1006 2.5 1006 Intraluminal pressure (mmHg) Stress (dynes cm-2) (A) (C) (B) (D) Figure 2 (A) (C) (B) (D) Figure 3 020 40 60 80 100 120 100 150 200 250 300 350 WT HFD WT SD WT VOO WT OT Intraluminal pressure (mmHg) Internal diameter (m) (1.8 mmol/L Ca2+) 020 40 60 80 100 120 100 150 200 250 300 350 ApoE HFD ApoE VOO ApoE OT ApoE SD * # # Intraluminal pressure (mmHg) Internal diameter (m) (1.8 mmol/L Ca2+) 020 40 60 80 100 120 50 60 70 80 90 100 110 * * Intraluminal pressure (mmHg) DiCa / Di0Ca (%) 020 40 60 80 100 120 50 60 70 80 90 100 110 * # # Intraluminal pressure (mmHg) DiCa / Di0Ca (%) (A) (C) (B) (D) Figure 4 -9 -8 -7 -6 -5 -4 0 25 50 75 100 ApoE SD ApoE HFD ApoE VOO ApoE OT ** ## ****** ## ## ## ## Log [ACh] % Relaxation -9 -8 -7 -6 -5 -4 0 20 40 60 80 L-NAMEL-NAME # * Log [ACh] % Relaxation -9 -8 -7 -6 -5 -4 0 20 40 60 80 Indo + L-NAME # Log [ACh] % Relaxation SD HFD VOO OT 0 100 200 300 * # AUC (A.U.)