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Functional foods and nutraceutical products for the management of cardiovascular alterations associated to aging and obesity
Spain (IFMSA-SPAIN)-University Autonoma of Madrid, Madrid
Physiology Department, School of Medicine, Universidad Autónoma de Madrid. Arzobispo Morcillo Street, 4, 28029 Madrid
Miriam Granado García/Angel Luis García Villalón
Miriam Granado García/Angel Luis García Villalón
Required: English, Accepted: Spanish
Type of Research Project
- Basic science
What is the background of the project?
In recent years, the progressive aging of the population has resulted in a high incidence of metabolic and cardiovascular diseases among the elderly population. The incidence of these diseases is largely related to unhealthy lifestyle habits among which sedentary life and hypercaloric diets rich in sugars and saturated fats are found. Several studies have shown that the intake of omega-3 fatty acids (PUFAs ω-3) improves the lipid profile, insulin sensitivity and cardiovascular disorders associated with obesity or aging. However, in most of these studies, FA-3 PUFAs from fish oils have been used, whose production is not sustainable in the long term. The seaweed oil, rich in PUFAs ω-3 (docosahexaenoic acid (DHA) mainly), is a low cost alternative to obtain FA-3 PUFAs, however, due to its high susceptibility to oxidation, at the moment, it is little used .
What is the aim of the project?
The aim of the project is to develop a new functional ingredient based in olive and algae oils useful for the treatment/prevention of the cardiometabolic alterations associated to aging.
What techniques and methods are used?
Organ bath for vascular reactivity experiments Langendorff (heart perfusion) for cardiac function Western Blot for protein quantification and quantitative polymerase chain reaction (qPCR) for gene expression analysis 1. Vascular reactivity experiments: After sacrificing the animals, the aortas will be dissected, cut into segments of 2 mm and mounted in an organ bath chamber full with Krebs-Henseleit physiological solution at 37ºC (4ml). The setting process will consist of introducing two steel wires through the light of the segments, one of each is fixed to the chamber and the other one is connected to a transducer to record changes in isometric tension. After stretching the aorta segments to an optimal passive tension (1g) and equilibrating them for 1h, KCL (100 mM) is added to the organ bath to test the functionality of the segments. Segments contracting less than 0.5g in response to KCl are discarded. After removing KCl from the chambers, segments will be precontracted with the thromboxane analogue U-46619 (10-7M). In the precontracted segments we will study the endothelium-dependent relaxation adding increasing doses of acetylcholine (10-10-10-4M) in the presence or absence of the nitric oxide synthesis blocker L-NAME (10-4 M). 2. Heart perfusion (Langendorff): After sacrifice, the heart will be removed immediately and cannulated through the aorta. The heart will be introduced in a chamber at 37ºC of temperature and the coronary circulation will be perfused retrogradely with physiological Krebs-Henseleit solution at a constant flow between 11-15 ml/min for the rat heart and between 3-7 ml/min for the mice heart using a peristaltic pump. Hearts (n=6) will undergo 30 min of ischemia by stopping the infusion pump followed by 45 minutes of reperfusion (IR). Immediately after the above experimental procedures, changes in coronary perfusion pressure, heart rate and ventricular contractility (determined by intraventricular pressure and dP/dt) will be recorded. The coronary perfusion pressure will be measured by a transducer connected proximally to the cannula and intraventricular pressure will be measured through a latex balloon inserted into the left ventricle connected to another transducer. Heart rate and heart contractility (dp/dt) will be calculated from intraventricular pressure. 3.Protein analysis by Western Blot: 100mg of myocardial/arterial tissue is homgenized using RIPA buffer, and total protein content is analyzed by the Bradford method (Bradford 1976). For each assay, resolving gels with SDS acrylamide (8-12%) are used and 100 μg of protein are loaded in each well. After electrophoresis, proteins are transferred to polyvinylidine difluoride (PVDF) membranes (Bio-Rad, Hércules, CA, USA) and transfer efficiency is determined by Ponceau red dyeing. Membranes are then blocked with Tris-buffered saline (TBS) containing 5% (w/v) non-fat dried milk and incubated with the appropriate primary antibody. Membranes are subsequently washed and incubated with the corresponding secondary antibody conjugated with peroxidase (1:2000; Pierce, Rockford, IL, USA). Peroxidase activity is visualized by chemiluminescence and quantified by densitometry using BioRad Molecular Imager ChemiDoc XRS System. All data are normalized to the housekeeping protein GAPDH (ThermoFisher) and referred to % of control values (non-ischemic hearts) on each gel. 4. mRNA analysis by quantitative real time PCR (qRT-PCR: a) RNA preparation and purification Total RNA is extracted from myocardial and arterial tissue according to the Tri-Reagent protocol (Chomczynski, 1993) and quantified with Nanodrop 2000 (ThermoScientific). cDNA is then synthesized from 1 µg of total RNA using a high capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). b) Quantitative real-time PCR The gene expression of different genes related to inflammation and oxidative stress assessed in myocardial and arterial tissue by quantitative real-time PCR. Quantitative real-time PCR is performed using assay-on-demand kits (Applied Biosystems). TaqMan Universal PCR Master Mix (Applied Biosystems) was used for amplification according to the manufacturer’s protocol in a Step One machine (Applied Biosystems). Values were normalized to the housekeeping gene 18S. According to manufacturer’s guidelines, the ∆∆CT method was used to determine relative expression levels. Statistics were performed using ∆∆CT values (Livak & Schmittgen, 2001). Statistical analysis Values are expressed as means ± S.E.M., and analyzed by one-way ANOVA followed by Bonferroni post-hoc test. A P value of <0.05 was considered significant.
What is the role of the student?
- The student will observe the practical experiments but will be highly involved in the analysis of the results
What are the tasks expected to be accomplished by the student?
To be incorporated and to learn the basic dymanics within a laboratory, materials, facilities and members. To learn different lab techniques such as Western Blot, qPCR and organ bath for the assessment of vascular and cardiac function. To properly handle the material and do the techniques by themselves, always assisted by a professional. To assist to scientific meetings to discuss the results
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- No specific outcome is expected
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
Knowledge of basic physiology of the cardiovascular system.
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts: - Medical students - Pre-Medical students from the American-British system - Students in biomedical fields
- 2019. Amor S; González-Hedström D; Martín-Carro B; Inarejos-García AM; Almodóvar P; Prodanov M; García-Villalón AL; Granado M. Beneficial effects of an aged black garlic extract in the metabolic and vascular alterations induced by a high fat/sucrose diet in male rats. Nutrients. 2019 Jan 12;11(1). pii: E153. doi: 10.3390/nu11010153.
- 2019. Miriam Granado; Sara Amor; Beatriz Martín-Carro; Lucía Guerra-Menéndez; Daniel Gonzalez-Hedström; Carmen Rubio; Jose Mª Carrascosa; Ángel Luis García-Villalón.Caloric restriction attenuates aging-induced cardiac insulin resistance in male Wistar rats through activation of PI3K/Akt pathway. Nutr Metab Cardiovasc Dis. 2019 Jan;29(1):97-105. doi: 10.1016/j.numecd.2018.09.005.
- 2018. Amor S; Martín-Carro B; Rubio C; Carrascosa JM; Hu W; Huang Y; García-Villalón AL; Granado M. Study of insulin vascular sensitivity in aortic rings and endothelial cells from aged rats subjected to caloric restriction: Role of perivascular adipose tissue. Experimental Gerontology pii: S0531-5565(17)30591-0. doi: 10.1016/j.exger.2017.10.017. (Q1)
- 2017. Granado M; Amor S; Fernández N; Carreño-Tarragona G; Martín-Carro B; Monge L; García-Villalón AL. Effects of early overnutrition on the renal response to Angiotensin II and expression of RAAS components in rat renal tissue. Nutrition; Metabolism and Cardiovascular Diseases. 2017 Oct;27(10):930-937. doi: 10.1016/j.numecd.2017.06.019 (Q1)
- 2017. Amor S; García-Villalón AL; Rubio C; Carrascosa JM; Monge L; Fernández N; Granado M. Effects of age and caloric restriction in the vascular response of renal arteries to endothelin-1 in rats. Experimental Gerontology. Feb;88:32-41. doi: 10.1016/j.exger.2016.12.020. (Q1)
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