1. Investigación

Most of the drugs used to treat obstructive pulmonary diseases are administered by the inhalation route. However, an incorrect inhalation technique may compromise the control of these pathologies. Objectives: The aim of this study was to evaluate the inhalation technique on patients of a community pharmacy and their medication adherence. Methods: A descriptive, cross-sectional observational study was carried out with 59 patients treated with inhalers who were surveyed on their inhalation technique and adherence to treatment. In addition, they were asked for a practical demonstration of their inhalation technique. Results: 47% of the surveyed patients committed critical inhaler use errors despite the fact that 97.3% of them considered they knew how to use properly their devices. The most frequent error was not to wait at least one minute between inhalations when it was necessary to repeat doses. Although 96% of the surveyed patients assured to have received information about how to use the inhaler, only 1% was provided by pharmacists. 47% of the patients showed a good adherence to the treatment. Nevertheless, 60% showed some type of non-compliance, being the erratic non-compliance the most frequent one. Conclusion: This study shows that a high percentage of patients present an incorrect inhalation technique and have a poor therapeutic adherence, which can contribute to a lower effectiveness of the treatment and a worse control of their health problems. A more active role of community pharmacists is needed to prevent this situation.

Aim: In addition to functioning as an energy sensor switch, AMPK plays a key role in the maintenance of cardiovascular homeostasis. However, obesity disrupts AMPK signaling, contributing to endothelial dysfunction and cardiovascular disease. This study aimed to elucidate if a short-term dietary intervention consisting in replacing the high-fat diet with a standard diet for 2 weeks could reverse obesity-induced endothelial dysfunction via AMPK-CREB activation. Methods: For this, 5-week-old male C57BL6J mice were fed a standard (Chow) or a high-fat (HF) diet for 8 weeks. The HF diet was replaced by the chow diet for the last 2 weeks in half of HF mice, generating 3 groups: Chow, HF and HF-Chow. Vascular reactivity and western-blot assays were performed in the thoracic aorta. Results: Returning to a chow diet significantly reduced body weight and glucose intolerance. Relaxant responses to acetylcholine and the AMPK activator (AICAR) were significantly impaired in HF mice but improved in HF-Chow mice. The protein levels of AMPKα, p-CREB and antioxidant systems (heme oxygenase-1 (HO-1) and catalase) were significantly reduced in HF but normalized in HF-Chow mice. Conclusion: Improving dietary intake by replacing a HF diet with a standard diet improves AMPK-mediated responses due to the upregulation of the AMPK/ CREB/HO-1 signaling pathway.

The non-steroidal mineralocorticoid receptor antagonist (MRA) finerenone (FIN) improves kidney and cardiovascular outcomes in patients with chronic kidney disease (CKD) in type 2 diabetes (T2D). We explored the effect of FIN in a novel model of type 1 diabetic Munich Wistar Fr¨omter (MWF) rat (D) induced by injection of streptozotocin (15 mg/kg) and additional exposure to a high-fat/high-sucrose diet. Oral treatment with FIN (10 mg/kg/day in rat chow) in diabetic animals (D-FIN) was compared to a group of D rats receiving no treatment and a group of non-diabetic untreated MWF rats (C) (n = 7–10 animals per group). After 6 weeks, D and D-FIN exhibited significantly elevated blood glucose levels (271.7 ± 67.1 mg/dl and 266.3 ± 46.8 mg/dl) as compared to C (110.3 ± 4.4 mg/dl; p < 0.05). D showed a 10-fold increase of kidney damage markers Kim-1 and Ngal which was significantly suppressed in D-FIN. Blood pressure, pulse wave velocity (PWV) and arterial collagen deposition were lower in D-FIN, associated to an improvement in endothelial function due to a reduction in procontractile prostaglandins, as well as reactive oxygen species (ROS) and inflammatory cytokines (IL-1, IL-6, TNFα and TGFβ) in perivascular and perirenal adipose tissue (PVAT and PRAT, respectively). In addition, FIN restored the imbalance observed in CKD between the procalcifying BMP-2 and the nephroprotective BMP-7 in plasma, kidney, PVAT, and PRAT. Our data show that treatment with FIN improves kidney and vascular damage in a new rat model of DKD with T1D associated with a reduction in inflammation, fibrosis and osteogenic factors independently from changes in glucose homeostasis.

Compound 21 (C21), a selective agonist of angiotensin type 2 receptor (AT2R), induces vasodilation through NO release. Since AT2R seems to be overexpressed in obesity, we hypothesize that C21 prevents the development of obesity-related vascular alterations. The main goal of this study was to assess the effect of C21on thoracic aorta endothelial function in a model of diet-induced obesity and to elucidate the potential crosstalk between AT2R, MasR and/or B2R in this response. 5-week-old male C57BL6J mice were fed a standard (CHOW) or a high-fat diet (HF) for 6 weeks and treated daily with C21 (1mg/Kg p.o) or vehicle, generating four groups: CHOW-C, CHOW-C21, HF-C, HF-C21. Vascular reactivity experiments were performed in thoracic aorta rings. Human endothelial cells (EA.hy926) were used to elucidate the signaling pathways, both at receptor and intracellular levels. Arteries from HF mice exhibited increased contractions to Ang II than CHOW mice, effect that was prevented by C21. PD123177, A779 and HOE-140 (AT2R, Mas and B2R antagonists) significantly enhanced Ang II-induced contractions in CHOW but not in HF-C rings, suggesting a lack of functionality of those receptors in obesity. C21 prevented those alterations and favoured the formation of AT2R/MasR and MasR/B2R heterodimers. HF mice also exhibited impaired relaxations to acetylcholine due to a reduced NO availability. C21 preserved NO release through PKA/p-eNOS and AKT/peNOS signaling pathways. In conclusion, C21 favours the interaction between AT2R, MasR and B2R and prevents the development of obesity-induced endothelial dysfunction by stimulating NO release through PKA/p-eNOS and AKT/p-eNOS signaling pathways.

The aim of this study was to evaluate the effect of Compound 21 (C21), a selective AT2R agonist, on the prevention of endothelial dysfunction, extracellular matrix (ECM) remodeling and arterial stiffness associated with diet-induced obesity (DIO). 5-week-old male C57BL/6J mice were fed a standard (Chow) or high-fat diet (HF) for 6 weeks.Half of the animals of each group were simultaneously treated with C21 (1mg/kg/day, in the drinking water), generating 4 groups: Chow C, Chow C21, HF C, HF C21. Vascular function and mechanical properties were determined in the abdominal aorta. To evaluate ECM remodeling, collagen deposition, activity of metalloproteinases (MMP) 2 and 9 and TGF-1 concentration were analyzed in the plasma. Abdominal aortas from HF C mice showed endothelial dysfunction as well as enhanced contractile but reduced relaxant responses to Ang II.This effect was abrogated with C21 treatment by preserving NO availability. A left-shift inthe tension-stretch relationship, paralleled by an augmented β-index (marker of intrinsic arterial stiffness), and enhanced collagen deposition and MMP-2/-9 activities were also detected in HF mice. However, when treated with C21, HF mice exhibited lower TGF-1 levels in abdominal aortas together with reduced MMP activities and collagen deposition compared with HF C mice. In conclusion, these data demonstrate that AT2R stimulation by C21 in obesity preserves NO availability and prevents unhealthy vascular remodeling, thus protecting the abdominal aorta in HF mice against the development of endothelial dysfunction, ECM remodeling and arterial stiffness.

The Munich Wistar Frömter (MWF) rat strain represents an experimental model to study cardiovascular alterations under conditions of progressive albuminuria. The aim of this study was to evaluate the association between genetic predisposition to albuminuria and the development of arterial stiffness and/or vascular remodelling. Experiments were performed in mesenteric arteries from 12-week-old MWF, Wistar Kyoto (WKY) and consomic MWF-6SHR and MWF-8SHR rats in which chromosomes 6 or 8 associated with albuminuria from MWF were replaced by the respective chromosome from spontaneously hypertensive rats (SHR). Incremental distensibility, wall stress and strain were reduced, and arterial stiffness was significantly increased in albuminuric MWF compared with WKY. Albuminuria suppression in both consomic strains was associated with lower β-values in MWF-8SHR and MWF-6SHR compared with MWF. Moreover, elastin content was significantly lower in MWF external elastic lamina compared with WKY and both consomic strains. In addition, a reduction in arterial external and internal diameter and cross-sectional area was detected in MWF compared with WKY, thus exhibiting an inward hypotrophic remodelling. However, these alterations remained unchanged in both consomic strains. These data demonstrate that albuminuria in MWF is associated with increased arterial stiffness due to a reduction of elastin content in the external elastic lamina. Moreover, inward hypotrophic remodelling in MWF is not directly associated with albuminuria. In contrast, we demonstrated that two major genetic loci affect both the development of albuminuria and arterial stiffness, thus linking albuminuria and impairment of mechanical properties of resistance arteries.

Perivascular adipose tissue (PVAT) releases numerous factors and adipokines with paracrine effects on both vascular structure and function. These effects are variable as they depend on regional differences in PVAT among blood vessels and vary with changes in adiposity. There is considerable evidence demonstrating an association between coronary PVAT and the development and progression of coronary artery disease, which is associated with inflammation, oxidative stress, angiogenesis, vascular remodelling and blood clotting. However, PVAT also has a protective role in vascular grafts, especially the no-touch saphenous vein, in patients undergoing coronary artery bypass. This beneficial influence of PVAT involves factors such as adipocyte-derived relaxing factor, nitric oxide (NO), leptin, adiponectin, prostanoids, hydrogen sulphide and neurotransmitters, as well as mechanical protection. This article aims to highlight and compare the dual role of PVAT in the development and progression of coronary atherosclerosis, as well as in increased graft patency. Different deleterious and protective mechanisms of PVAT are also discussed and the inside-outside signalling paradigm of atherosclerosis development re-evaluated. The bidirectional communication between the arterial and venous wall and their surrounding PVAT, where signals originating from the vascular wall or lumen can affect PVAT phenotype, has been shown to be very complex. Moreover, signals from PVAT also influence the structure and function of the vascular wall in a paracrine manner.

Dietary treatment with high-fat diets (HFD) triggeenrs diabetes and hyperleptinemia, con- comitantly with a partial state of leptin resistance that affects hepatic and adipose tissue but not the heart. In this context, characterized by widespread steatosis, cardiac lipid con- tent remains unchanged. As previously reported, HFD-evoked hyperleptinemia could be a pivotal element contributing to increase fatty-acid (FA) metabolism in the heart and to prevent cardiac steatosis. This metabolic adaptation might theoretically reduce energy efficiency in cardiomyocytes and lead to cardiac electrophysiological remodeling. There- fore the aim of the current study has been to investigate the impact of long-term HFD on cardiac metabolism and electrophysiological properties of the principal ionic currents responsible of the action potential duration in mouse cardiomyocytes. Male C57BL/6J mice were fed a control (10 kcal% from fat) or HFD (45 kcal% from fat) during 32 weeks. Quantification of enzymatic activities regulating mitochondrial uptake of pyruvate and FA showed an increase of both carnitine-palmitoyltransferase and citrate synthase activities together with a decrease of lactate dehydrogenase and pyruvate dehydrogenase activities. Increased expression of uncoupling protein-3, Mn-, and Cu/Zn-superoxide dismutases and catalase were also detected. Total glutathione/oxidized glutathione ratios were unaffected by HFD. These data suggest that HFD triggers adaptive mechanisms aimed at facilitating FA catabolism, and preventing oxidative stress. All these changes did not affect the dura- tion of action potentials in cardiomyocytes and only slightly modified electrocardiographic parameters.

Leptin causes vasodilatation both by endothelium-dependent and-independent mechanisms. Leptin is synthesized by perivascular adipose tissue (PVAT). The hypothesis of this study is that a decrease of leptin production in PVAT of spontaneously hypertensive rats (SHR) might contribute to adiminished paracrine anticontractile effect of the hormone. We have determined in aorta from Wistar-Kyoto (WKY) and SHR leptin mRNA and protein levels in PVAT, the effect of leptin and PVAT on contractile responses, and leptin-induced relaxation and nitricoxide (NO) production. Leptin mRNA and protein expression were significantly lower in PVAT from SHR. Concentration response curves to angiotensin II were significantly blunted in presence of PVAT as well as by exogenousleptin (10−9M) only in WKY. This anticontractile effect was endothelium-dependent. Vasodilatation induced by leptin was smaller in SHR than in WKY, and was also endothelium-dependent. More over, release of endothelial NO in response to acute leptin was higherin WKY compared to SHR, but completely abolished in the absence of endothelium. In conclusion, the reduced anticontractile effect of PVAT in SHR might be attributed to a reduced PVAT-derived leptin and to an abrogated effect of leptin on endothelial NO release probably due to an impaired activation of endothelial NO synthase

Background: The hypothesis of this study is that long-term high-fat diets (HFD) induce perivascular adipose tissue (PVAT) dysfunction characterized by a redox imbalance, which might contribute to aggravate endothelial dysfunction in obesity. Methods and Results: C57BL/6J mice were fed either control or HFD (45% kcal from fat) for 32 weeks. Body weight, lumbar and mesenteric adipose tissue weights were significantly higher in HFD animals compared to controls. The anticontractile effect of PVAT in mesenteric arteries (MA) was lost after 32 week HFD and mesenteric endothelial-dependent relaxation was significantly impaired in presence of PVAT in HFD mice (Emax = 71.065.1 vs Emax = 58.564.2, p,0.001). The inhibitory effect of L-NAME on Ach-induced relaxation was less intense in the HFD group compared with controls suggesting a reduction of endothelial NO availability. Expression of eNOS and NO bioavailability were reduced in MA and almost undetectable in mesenteric PVAT of the HFD group. Superoxide levels and NOX activity were higher in PVAT of HFD mice. Apocynin only reduced contractile responses to NA in HFD animals. Expression of ec-SOD and total SOD activity were significantly reduced in PVAT of HFD mice. No changes were observed in Mn-SOD, Cu/Zn-SOD or catalase. The ratio [GSSG]/([GSH]+[GSSG]) was 2- fold higher in the mesenteric PVAT from HFD animals compared to controls. Conclusions: We suggest that the imbalance between pro-oxidant (NOX, superoxide anions, hydrogen peroxide) and antioxidant (eNOS, NO, ecSOD, GSSG) mechanisms in PVAT after long-term HFD might contribute to the aggravation of endothelial dysfunction.