Mecanismos implicados en los cambios estructurales, mecánicos y funcionales asociados a procesos inflamatorios de arterias cerebrales y mesentéricas de resistencia de rata

Consultable des del TDXTítol obtingut de la portada digitalitzadaEl objetivo de esta tesis ha sido estudiar y analizar las modificaciones estructurales, mecánicas y funcionales que se producen en un proceso inflamatorio en general y en concreto en un episodio de isquemia cerebral. El trabajo realizado se compone de dos partes: i) estudio del efecto de un proceso de isquemia-reperfusión sobre las propiedades estructurales, mecánicas y miogénicas en la arteria cerebral media de rata. ii) estudio mediante un modelo «in vitro» de inflamación que consiste en la incubación de las arterias en medio de cultivo con una de las citoquinas (IL-1ß) que se sobrexpresan después de un período de isquemia-reperfusión, sobre las propiedades estructurales, mecánicas y funcionales de la arteria mesentérica de resistencia de rata. El proceso de isquemia (90 minutos)-reperfusión (24 horas) produjo los siguientes cambios en la arteria cerebral media: 1) disminución importante del tono miogénico acompañado por un aumento en la producción de aniones superóxido 2) incremento en el tamaño de la pared vascular debido al aumento en el número de células adventicias 3) aumento en el tamaño de las fenestras de la lámina elástica interna, hecho que podría asociarse al aumento en la elasticidad intrínseca observado en estas arterias 4) disminución del estrés de la pared como consecuencia del aumento en el tamaño de la misma 5) este proceso también modificó aunque con menor intensidad, algunos de los parámetros estructurales analizados en arterias del lado contralateral . Estos cambios, se deban probablemente a alteraciones sistémicas consecuencia de la isquemia focal. La incubación de las arterias mesentéricas de resistencia en medio de cultivo con IL-1ß durante 14 horas: 1) no modificó ni la estructura ni la mecánica de la pared arterial 2) empeoró las relajaciones dependientes de óxido nítrico sin afectar a la capacidad vasorelajadora general del vaso. El empeoramiento de la relajación parece ser debido, al menos en parte, a una disminución en la biodisponibilidad del óxido nítrico 3) aumentó la producción de anión superóxido en la pared vascular que parecería estar sintetizado principalmente por la xantina oxidasa. Los aniones superóxido producidos por acción de la xantina oxidasa serían los principales responsables de la disminución en la biodisponibilidad del óxido nítrico, que a su vez sería el responsable de la disminución de las respuestas relajantes mediadas por óxido nítrico. Estos resultados indicarían que la arteria cerebral media sufre una serie de cambios provocados por el proceso de isquemia-reperfusión que permitirían a estas arterias adaptarse a los cambios en el flujo sanguíneo necesarios para reducir el daño neuronal, mejorando la irrigación del cerebro después de un episodio de isquemia-reperfusión. El efecto de las citoquinas durante este período podría también afectar a las respuestas funcionales de las arterias debido al aumento en la producción de anión superóxido observado en esta patología. Las aportaciones de estos estudios podrían en un futuro abrir líneas de investigación que podrían contribuir al diseño de nuevos fármacos con capacidad para prevenir episodios de isquemia cerebral.The aim of the present study has been to analyse the structural, mechanical and functional alterations that take place in an inflammatory process as cerebral ischaemia. Our results are divided in two parts: i) a study of the effect of ischaemia-reperfusion on structural, mechanical and myogenic properties of rat middle cerebral artery and ii) to analyze, using an in vitro model of inflammation, the effect of incubation with IL-1ß, one of several cytokines that is overexpressed after a period of ischemia-reperfusion, on structural mechanical and functional properties of rat mesenteric resistance arteries. The occlusion of the right middle cerebral artery (90 minuts) following 24 hours of reperfusion produced: 1) a decrease of myogenic tone together with an increase in superoxide anion production 2) an enlargement of the vascular wall due to the increase in the total number of adventitial cells 3) an enlargement of fenestrae area of the internal elastic lamina that was associated with the increase on intrinsic elasticity observed in these arteries 4) a diminished wall stress as a result of the increase in the wall thickness 5) ischaemia-reperfusion also modified although to a lesser degree, some of the structural parameters in arteries from the contralateral side of ischemia. These changes observed in the contralateral side could likely be associated to systemic alterations produced as a consequence of focal ischaemia. Fourteen hours incubation of mesenteric resistance arteries in culture medium with IL-1ß: 1) did not modify either the structure or the mechanics of the arterial wall; 2) impaired nitric oxide dependent relaxation without affecting the vasodilatation capacity of the arteries. The impairment of nitric oxide induced relaxation by IL-1ß could be due to a decrease on nitric oxide availability; 3) increased superoxide anion production in the vascular wall that seems to be mainly synthesized by xanthine oxidase. The superoxide anion produced by xanthine oxidase would play a role in decreasing nitric oxide availability, which in turn would diminish vasodilatation mediated by nitric oxide. These results suggest that the middle cerebral artery undergoes multiple changes caused by ischaemia-reperfusion that will allow these arteries to adapt to the blood flow alterations needed to reduce the neural damage, improving brain blood flow. Cytokines could affect functional responses of the arteries due to the increase in superoxide anion production observed in this pathology. In the future, this study could open new research that could contribute to the design of new drugs with potential interest to prevent cerebral ischaemia episodes

Uric acid therapy for vasculoprotection in acute ischemic stroke

Uric acid (UA) is a product of the catabolism of purine nucleotides, the principal constituents of DNA, RNA, and cellular energy stores, such as adenosine triphosphate. The main properties of UA include scavenging of hydroxyl radicals, superoxide anion, hydrogen peroxide, and peroxynitrite that make this compound to be the most potent antioxidant in the human plasma. As the result of two silencing mutations in the gene of the hepatic enzyme uricase which degrades UA to allantoin, humans have higher levels of UA than most mammals. However, these levels rapidly decrease following an acute ischemic stroke (AIS), and this decrement has been associated to worse stroke outcomes. This review highlights the safety and potential clinical value of UA therapy in AIS, particularly in patients more exposed to redox-mediated mechanism following the onset of ischemia, such as women, hyperglycemic patients, or patients treated with mechanical thrombectomy. The clinical findings are supported by preclinical data gathered in different laboratories, and in assorted animal species which include male and female individuals or animals harboring comorbidities frequently encountered in patients with AIS, such as hyperglycemia or hypertension. A remarkable finding in these studies is that UA targets its main effects in the brain vasculature since available evidence suggests that does not seem to cross the blood-brain barrier. Altogether, the available data with UA therapy extend the importance of vasculoprotection for effective neuroprotection at the bedside and reinforce the role of endothelial cells after brain ischemia for an increased survival of the whole neurovascular unit

Reactive Oxygen Species and Oxidative Stress in the Pathogenesis and Progression of Genetic Diseases of the Connective Tissue

Connective tissue is known to provide structural and functional 'glue' properties to other tissues. It contains cellular and molecular components that are arranged in several dynamic organizations. Connective tissue is the focus of numerous genetic and nongenetic diseases. Genetic diseases of the connective tissue are minority or rare, but no less important than the nongenetic diseases. Here we review the impact of reactive oxygen species (ROS) and oxidative stress on the onset and/or progression of diseases that directly affect connective tissue and have a genetic origin. It is important to consider that ROS and oxidative stress are not synonymous, although they are often closely linked. In a normal range, ROS have a relevant physiological role, whose levels result from a fine balance between ROS producers and ROS scavenge enzymatic systems. However, pathology arises or worsens when such balance is lost, like when ROS production is abnormally and constantly high and/or when ROS scavenge (enzymatic) systems are impaired. These concepts apply to numerous diseases, and connective tissue is no exception. We have organized this review around the two basic structural molecular components of connective tissue: The ground substance and fibers (collagen and elastic fibers)

Molecular Mechanisms Underlying the Effects of Olive Oil Triterpenic Acids in Obesity and Related Diseases

Dietary components exert protective effects against obesity and related metabolic and cardiovascular disturbances by interfering with the molecular pathways leading to these pathologies. Dietary biomolecules are currently promising strategies to help in the management of obesity and metabolic syndrome, which are still unmet medical issues. Olive oil, a key component of the Mediterranean diet, provides an exceptional lipid matrix highly rich in bioactive molecules. Among them, the pentacyclic triterpenic acids (i.e., oleanolic acid) have gained clinical relevance in the last decade due to their wide range of biological actions, particularly in terms of vascular function, obesity and insulin resistance. Considering the promising effects of these triterpenic compounds as nutraceuticals and components of functional foods against obesity and associated complications, the aim of our review is to decipher and discuss the main molecular mechanisms underlying these effects driven by olive oil triterpenes, in particular by oleanolic acid. Special attention is paid to their signaling and targets related to glucose and insulin homeostasis, lipid metabolism, adiposity and cardiovascular dysfunction in obesity. Our study is aimed at providing a better understanding of the impact of dietary components of olive oil in the long-term management of obesity and metabolic syndrome in humans

Vascular aging : facts and factors

"Man is as old as his arteries". This old aphorism has been widely confirmed by epidemiological and observational studies establishing that cardiovascular diseases can be age-related in terms of their onset and progression. Besides, with aging come a number of physiological and morphological changes that alters cardiovascular function and lead to subsequently increased risk of cardiovascular disease, even in health asymptomatic individuals. Even though different adaptive mechanisms to protect blood vessels against mild stress have been described, the aging process induces a progressive failure of protective mechanisms, leading to vascular changes. The outcomes of the aging-related modifications are the impairment of homeostasis of the irrigated organs and resultant target organ damage

Sex-Dependent End-of-Life Mental and Vascular Scenarios for Compensatory Mechanisms in Mice with Normal and AD-Neurodegenerative Aging

Life expectancy decreases with aging, with cardiovascular, mental health, and neurodegenerative disorders strongly contributing to the total disability-adjusted life years. Interestingly, the morbidity/mortality paradox points to females having a worse healthy life expectancy. Since bidirectional interactions between cardiovascular and Alzheimer's diseases (AD) have been reported, the study of this emerging field is promising. In the present work, we further explored the cardiovascular-brain interactions in mice survivors of two cohorts of non-transgenic and 3xTg-AD mice, including both sexes, to investigate the frailty/survival through their life span. Survival, monitored from birth, showed exceptionally worse mortality rates in females than males, independently of the genotype. This mortality selection provided a "survivors" cohort that could unveil brain-cardiovascular interaction mechanisms relevant for normal and neurodegenerative aging processes restricted to long-lived animals. The results show sex-dependent distinct physical (worse in 3xTg-AD males), neuropsychiatric-like and cognitive phenotypes (worse in 3xTg-AD females), and hypothalamic-pituitary-adrenal (HPA) axis activation (higher in females), with higher cerebral blood flow and improved cardiovascular phenotype in 3xTg-AD female mice survivors. The present study provides an experimental scenario to study the suggested potential compensatory hemodynamic mechanisms in end-of-life dementia, which is sex-dependent and can be a target for pharmacological and non-pharmacological interventions

Transient Mesenteric Ischemia Leads to Remodeling of Rat Mesenteric Resistance Arteries

Mesenteric ischemia/reperfusion (I/R) is associated with high rates of morbidity and mortality. We studied the effect of mesenteric I/R on structural and mechanical properties of rat mesenteric resistance artery (MRA) that, once disrupted, might impact the outcome of this devastating clinical condition. Superior mesenteric artery from Wistar–Kyoto rats was occluded (90 min) and reperfused (24 h). The effect of tezosentan, a dual endothelin (ET)-receptor antagonist, was studied in ischemic (IO) and sham-operated (SO) animals. MRA structure and mechanics were assessed by pressure myography. Nuclei distribution, elastin content and organization, collagen I/III and ET-1 expression, ET-1 plasma levels, superoxide anion (O2⋅−) production, and mRNA levels of NAD(P)H-oxidase subunits were measured. To assess ET-1 effects on O2⋅− production, MRA from non-operated rats were incubated in culture medium with ET-1. Mesenteric I/R increased MRA wall thickness (P < 0.05) and cross-sectional area (P < 0.05) but decreased wall stiffness (P < 0.05). Arterial remodeling was paralleled by enhancement of: (i) collagen I/III expression (P < 0.01), ET-1 expression (P < 0.05), and O2⋅− formation (P < 0.01) in the vessel wall; (ii) number of internal elastic lamina (IEL) fenestrae (P < 0.05); and (iii) plasma levels of ET-1 (P < 0.05). Moreover, ET-1 increased O2⋅− (P < 0.05) production in cultured MRA. Tezosentan prevented hypertrophic remodeling and collagen I/III deposition, and enhanced O2⋅− production, but it did not affect the decreased wall stiffness after mesenteric I/R. These results indicate that 90 min occlusion/24 h reperfusion induces hypertrophic remodeling of MRA linked to ET-1-mediated increase of collagen and O2⋅−. Decreased stiffness may be associated with increased number of IEL fenestrae. The resulting MRA remodeling, initially adaptive, might become maladaptive contributing to the pathology and poor outcome of mesenteric I/R, and might be a valuable treatment target for mesenteric I/R

Differences in the thoracic aorta by region and sex in a murine model of Marfan Syndrome

Marfan syndrome (MFS) is a hereditary disorder of the connective tissue that causes life-threatening aortic aneurysm, which initiates at the aortic root and can progress into the ascending portion. However, analysis of ascending aorta reactivity in animal models of MFS has remained elusive. Epidemiologic evidence suggests that although MFS is equally prevalent in men and women, men are at a higher risk of aortic complications than non-pregnant women. Nevertheless, there is no experimental evidence to support this hypothesis. The aim of this study was to explore whether there are regional and sex differences in the thoracic aorta function of mice heterozygous for the fibrillin 1 (Fbn1) allele encoding a missense mutation (Fbn1C1039G/+), the most common class of mutation in MFS. Ascending and descending thoracic aorta reactivity was evaluated by wire myography. Ascending aorta mRNA and protein levels, and elastic fiber integrity were assessed by qRT-PCR, Western blotting, and Verhoeff-Van Gieson histological staining, respectively. MFS differently altered reactivity in the ascending and descending thoracic aorta by either increasing or decreasing phenylephrine contractions, respectively. When mice were separated by sex, contractions to phenylephrine increased progressively from 3 to 6 months of age in MFS ascending aortas of males, whereas contractions in females were unchanged. Endothelium-dependent relaxation was unaltered in the MFS ascending aorta of either sex; an effect related to augmented endothelium-dependent hyperpolarization-type dilations. In MFS males, the non-selective cyclooxygenase (COX) inhibitor indomethacin prevented the MFS-induced enhancement of phenylephrine contractions linked to increased COX-2 expression. In MFS mice of both sexes, the non-selective nitric oxide synthase inhibitor L-NAME revealed negative feedback of nitric oxide on phenylephrine contractions, which was associated with upregulation of eNOS in females. Finally, MFS ascending aortas showed a greater number of elastic fiber breaks than the wild-types, and males exhibited more breaks than females. These results show regional and sex differences in Fbn1C1039G/+ mice thoracic aorta contractility and aortic media injuries. The presence of more pronounced aortic alterations in male mice provides experimental evidence to support that male MFS patients are at increased risk of suffering aortic complications

An Imidazoline 2 Receptor Ligand Relaxes Mouse Aorta via Off-Target Mechanisms Resistant to Aging

Imidazoline receptors (IR) are classified into three receptor subtypes (I1R, I2R, and I3R) and previous studies showed that regulation of I2R signaling has neuroprotective potential. In order to know if I2R has a role in modulating vascular tone in health and disease, we evaluated the putative vasoactive effects of two recently synthesized I2R ligands, diethyl (1RS,3aSR,6aSR)-5- (3-chloro-4-fluorophenyl)-4,6-dioxo-1-phenyl-1,3a,4,5,6,6a-hexahydropyrrolo[3,4-c]pyrrole -1- phosphonate (B06) and diethyl [(1-(3-chloro-4-fluorobenzyl)-5,5-dimethyl-4-phenyl-4,5-dihydro- 1H-imidazol-4-yl]phosphonate] (MCR5). Thoracic aortas from Oncins France 1 (3- to 4-monthsold) and C57BL/6 (3- to 4- and 16- to 17-months-old mice) were mounted in tissue baths to measure isometric tension. In young mice of both strains, MCR5 induced greater relaxations than either B06 or the high-affinity I2R selective ligand 2-(2-benzofuranyl)-2-imidazoline (2-BFI), which evokedmarginal responses.MCR5 relaxations were independent of I2R, as IR ligands did not significantly affect them, involved activation of smoothmuscle KATP channels and inhibition of L-type voltage-gated Ca2+ channels, and were only slightly modulated by endothelium-derived nitric oxide (negatively) and prostacyclin (positively). Notably, despite the presence of endothelial dysfunction in old mice, MCR5 relaxations were preserved. In conclusion, the present study provides evidence against a functional contribution of I2R in the modulation of vascular tone in the mouse aorta. Moreover, the I2R ligand MCR5 is an endothelium-independent vasodilator that acts largely via I2R-independent pathways and is resistant to aging. We propose MCR5 as a candidate drug for the management of vascular disease in the elderly