Cuando nuestras arterias envejecen

The aging of our arteries represents an important cardiovascular risk factor, associated with the lack of functionality of blood vessels and a reduced response to different stimuli, such as the activation of angiogenesis. In fact, the age-related lack of angiogenic response conditions the maintenance of the physiological vascular network and the repair of damaged tissues after episodes of ischemia. The maintainance of a good cardiovascular health means keeping our arteries young, and to do this, the prevention of oxidative damage is fundamental, with food bioactive compounds playing a very important role.El envejecimiento de nuestras arterias supone un importante factor de riesgo cardiovascular, asociado a una falta de funcionalidad de los vasos sanguíneos y a una respuesta disminuida frente a diferentes estímulos, como la activación de la angiogénesis. De hecho, la falta de respuesta angiogénica asociada a la edad condiciona el mantenimiento de la red fisiológica vascular y la reparación de tejidos dañados tras episodios de isquemia. Mantener una buena salud cardiovascular pasa por mantener jóvenes nuestras arterias, y para ello, la prevención del daño oxidativo es fundamental, jugando un papel muy importante los compuestos bioactivos presentes en los alimentos

Identification and characterization of new anti-angiogenic compounds from natural sources

The inhibition of angiogenesis has attracted broad attention in the field of pharmacological research, not only for cancer, but for other angiogenesis dependent diseases including ophthalmic, cutaneous and inflammatory diseases, as well as a number of rare diseases. Our research group has characterized multiple new natural bioactive compounds with multitargeted antiangiogenic effects by employing a well-established set of in vitro, in vivo and ex vivo preclinical models of angiogenesis. Most of them have been isolated from plants and terrestrial microorganisms, mainly due to their higher availability and because their therapeutic effects had been previously known in folk traditional medicines. In vitro primary screening includes cell differentiation and toxicity and proliferation assays. Secondary screening involves several experiments to evaluate effects on adhesion, migration, invasion, apoptosis or cell cycle analysis, among others. Additionally, we perform a further molecular characterization analyzing possible signaling pathways that are affected to elucidate their mechanism of action. The characterization is completed with the ex vivo aortic ring assay, and in vivo assays, as CAM and zebrafish assays, to ensure the anti-angiogenic ability. As a fruit of the mentioned screening, a number of compounds with remarkable anti-angiogenic activity have been identified and characterized.Our experimental work is supported by grants BIO2014-56092-R (MINECO and FEDER) and P12-CTS-1507 (Andalusian Government and FEDER) and funds from group BIO-267 (Andalusian Government). The "CIBER de Enfermedades Raras" is an initiative from the ISCIII (Spain)]. This communicaction has the support of a travel grant "Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech"

Interplay between glucose and palmitate uptake in breast carcinoma in vitro

One of the most studied tumor cells lines in vitro is the breast carcinoma MDA-MB-231 cell line. Several studies have proved its glycolytic profile, namely known as the Warburg effect. Glutamine oxidation is also important for its metabolism. Nevertheless, the use of fatty acids for obtaining energy in these cells is still rising. Palmitic acid is the most common saturated fatty acid, containing sixteen carbons in its structure. However, the use of palmitate for metabolic studies in MDA-MB-231 is not very extended due to its pro-apoptotic effect in this cell line after certain time exposure. Nonetheless, in this work we used palmitate as a metabolic fuel for just 30 minutes in order to see the almost immediate response of the cells to its presence, after a 30 minutes fast period. Our results show that MDA-MB-231 cells are not able of oxidizing palmitate nor producing lactate from it. Simultaneous presence of palmitate with glucose or with glutamine does not affect glucose nor glutamine uptake in these cells. However, we observed that even low concentrations of glucose increase palmitate uptake in MDA-MB-231 after a 30 minutes incubation. Treatment with 5 mM 2-deoxyglucose also for 30 minutes counters this rise, since 2-deoxyglucose diminishes palmitate uptake. Increasing glucose concentration to the same dosis of 2-deoxyglucose leads to a prevalence of the glucose effect on palmitate uptake. The exact role of glucose and glucose derivatives should be further studied in order to know more about palmitate metabolism in this cell line.Our experimental work is supported by grants BIO2014-56092-R (MINECO and FEDER) and P12-CTS-1507 (Andalusian Government and FEDER) and funds from group BIO-267 (Andalusian Government). The "CIBER de Enfermedades Raras" is an initiative from the ISCIII (Spain). This communication has the support of a travel grant "Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech"

Fasentin, a glucose uptake inhibitor, is also able to inhibit angiogenesis

Es comunicación a congreso en formato pósterThe role of glucose on endothelial cell (EC) metabolism and angiogenesis has been an emerging issue in the last few years. Some inhibitors of glucose metabolism, such as 2-deoxyglucose, have been shown to have anti-angiogenic effects. Fasentin is a poor-studied inhibitor of glucose uptake which modulates GLUT-1 and GLUT-4 transporters in cancer cells. We wanted to test its possible effect on EC glucose uptake, showing a light decrease in HMEC at 100 µM. Lower doses did not affect this characteristic of glucose metabolism. In line with this fact, fasentin at 100 µM totally inhibited tube formation on Matrigel in these cells. This anti-angiogenic effect is not likely to be helped by a pro-apoptotic effect of fasentin but, as proved with additional assays, it could be due to a decrease on the signaling for extracellular matrix degradation. More research would be necessary in order to elucidate its fine regulation on angiogenesis and metabolism.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. [Our experimental work is supported by grants BIO2014-56092-R (MINECO and FEDER) and P12-CTS-1507 (Andalusian Government and FEDER) and funds from group BIO-267 (Andalusian Government). The "CIBER de Enfermedades Raras" is an initiative from the ISCIII (Spain). This communicaction has the support of a travel grant "Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech"]

Lactate Oxidation in Endothelial Cells: A Feature of All Endothelial Cells?

Resumen de la comunicaciónMetabolism of endothelial cells is a topic that has gained an increasing interest in the last years. This is due to their role in the angiogenic process, which is pathologically upregulated in several diseases, such as retinopathies, diabetes and cancer. Glycolysis, among other metabolic routes, has been found to be essential for triggering the angiogenic switch. Additionally, it has been seen that endothelial cells are able to take up lactate from the extracellular media, for example in the case of the tumor microenvironment, where cancer cells would have secreted high amounts of this metabolite. Endothelial cells would oxidize this lactate for obtaining energy, but lactate can also act as a signaling molecule for the angiogenic process. However, experiments to determine the molecular fate of lactate have been performed using only macrovascular endothelial cells. The aim of the present work is to prove whether microvascular endothelial cells are also able to take up and oxidize lactate. For this purpose, fluorimetry, isotopic labeling and Seahorse experiments were used to study the metabolism of a human microvascular endothelial cell line (HMEC). The expression levels of transcripts and proteins of different enzymes and transporters related to lactate metabolism were estimated by qPCR and Western blotting. The results obtained indicate that these cells rely on glycolysis for their metabolism, while the oxidation of glucose and glutamine seems to be considerably low. On the other hand, no lactate oxidation could be detected. We then checked the mRNA expression of the two isoenzymes of lactate dehydrogenase (LDH) and the two main lactate transporters, MCT1 and MCT4, and found that levels of LDH-B and MCT1 were undetectable. We failed to measure any MCT1 mRNA or protein expression either in normoxia or hypoxia. Hence, we can conclude that at least this microvascular endothelial cell line cannot use extracellular lactate as a metabolic fuel.Our experimental work is supported by grants BIO2014-56092-R (MINECO and FEDER) and P12-CTS-1507 (Andalusian Government and FEDER) and funds from group BIO-267 (Andalusian Government). The "CIBER de Enfermedades Raras" is an initiative from the ISCIII (Spain). This communicaction has the support of a travel grant "Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech"

The noni anthraquinone damnacanthal is a multi-kinase inhibitor with potent anti-angiogenic effects

Este es el manuscrito que fue aceptado y que finalmente se publicó en Cancer Letters con el DOI: 10.1016/j.canlet.2016.10.037The natural bioactive compound damnacanthal inhibits several tyrosine kinases. Herein, we show that -in fact- damancanthal is a multi kinase inhibitor. A docking and molecular dynamics simulation approach allows getting further insight on the inhibitory effect of damnacanthal on three different kinases: vascular endothelial growth factor receptor-2, c-Met and focal adhesion kinase. Several of the kinases targeted and inhibited by damnacanthal are involved in angiogenesis. Ex vivo and in vivo experiments clearly demonstrate that, indeed, damnacanthal is a very potent inhibitor of angiogenesis. A number of in vitro assays contribute to determine the specific effects of damnacanthal on each of the steps of the angiogenic process, including inhibition of tubulogenesis, endothelial cell proliferation, survival, migration and production of extracellular matrix remodeling enzyme. Taken altogether, these results suggest that damancanthal could have potential interest for the treatment of cancer and other angiogenesisdependent diseases.Supported by grants BIO2014-56092-R (MINECO and FEDER), P12-CTS-1507 (Andalusian Government and FEDER) and funds from group BIO-267 (Andalusian Government). The "CIBER de Enfermedades Raras" is an initiative from the ISCIII (Spain). JAGV had the financial support of Vicerrectorado de Investigación y Transferencia (University of Málaga, Spain). The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript

The enzymatic determination of glucose in carbonated beverages: a useful tool for the undergraduate students to learn the basis of enzymatic analysis and the comparison of two analytical methods

The importance of enzymatic analysis in biochemistry, clinical chemistry and food chemistry is undoubted. The course "Applied Biochemistry" in our Faculty is aimed to undergraduate students of Chemistry and Biochemistry. In this subject, the principles and applications of enzymatic analysis are presented to the students, who receive a theoretical introductory lecture in the classroom before they carry out an experiment that should be feasible to be solved in a short laboratory period. The experimental protocol here presented, based on the enzymatic determination of glucose in carbonated beverages, has been implemented at the University of Málaga and it has been optimized according to the students’ results and commentaries along the last years. It aims to illustrate basic issues relating enzymatic analysis, including its potential application to food chemistry. Although there are several enzymatic methods that can be used for the determination of glucose, we selected the one based on the coupled reactions of glucose oxidase (GOD; EC 1.1.3.4.) and peroxidase (POD; EC 1.11.1.7.) because the kinetic constants of glucose oxidase allow the mentioned enzymatic reactions to be used in both, the end point and the kinetic enzymatic analysis methods. In this way, data for two different protocols for the determination of glucose concentration are obtained by the students from a single reaction mixture. Students construct a calibration curve for each method using a glucose standard solution, and use them to determine the glucose concentration in the problem solutions. The inclusion of replicate samples in the determination of the glucose concentration of an “ideal problem” (glucose in purified water) is used to illustrate the principles of statistics in the lab, and comparison with the “real value” allows an estimation of the accuracy of each method. The evaluation of glucose concentration in four carbonated beverages: coloured coke and uncoloured tonic sodas (regular or sugarless in both cases) makes student to recognise the appearance of interferences that should be either avoided or eliminated. Since all samples are analysed by means of end-point and kinetic methods, students can discuss the applicability of each method to these specific analytical problems. They are also encouraged to compare both analytical methods in terms of sensitivity, selectivity, accuracy, and time consumed. Chemistry and Biochemistry undergraduate students having performed this experiment in our laboratories have found it formative, interesting and challenging.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

A practice project to prevent the cookbook model as modus operandi for biochemistry laboratory learning

Laboratory learning is a crucial component of chemistry and biochemistry instruction and should be conceived as a way to develop students’ reasoning, technical or practical skills, introducing them into the scientific method principles. Nevertheless, the heavily criticized “expository instruction style”, characterized by a cookbook nature, is still the most widespread style of laboratory instruction in our universities. Alternative learning styles based in the inquiry, discovery and problem-based pedagogical approaches, have been reported to promote students’ problem solving skills, critical thought and self-confidence development. We are currently involved in the Educative Innovation Project PIE17-065, funded by University of Malaga, aimed to improve the teaching practice of Biochemistry laboratory to undergraduate students. Based on an enzymatic analysis of glucose in soft-drinks we have developed a laboratory protocol as a part of a full practice project where students must work before and after the lab session, in order to prevent the cookbook model as modus operandi, therefore preventing the situation where the students get a first glimpse of the experiment protocol whereas they put on their lab coat. The learning activities have been designed to move our students from the passive role that characterizes the step-by-step procedures, to an active and critical attitude that starts before and remains after their laboratory session, also minimizing time, space, and equipment resources. Our results have shown that this experiment has improved the learning of both, future biochemists and chemists, which showed a very positive perception of the whole practical project.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. PIE 17-06

Synthesis and biological activity of a new class of antitumor cyclopeptides based on the solomonamides

Solomonamides A (1) and B (2) are novel natural products recently isolated from the marine sponge Theonella swinhoei [1]. Preliminary structural studies revealed an unprecedented cyclic peptide type structure. Interestingly, solomonamide A exhibits anti-inflammatory activity, showing potent reduction (60%) of inflammation at a very low concentration of 100 µg/kg in animal models. However, the scarcity of these compounds from their natural sources has been a drawback for further pharmacological assays. In fact, the anti-inflammatory activity of solomonamide B was not evaluated due to the limited amounts. This difficulty to access large amounts of these compounds makes quite difficult to gain insight into their biological profiles and mechanism of action and justifies the chemical synthesis of this new class of cyclic peptides. As a consequence, the solomonamides have been the subject of several synthetic efforts [2] notably by the Reddy group who has recently reported the first total synthesis of solomonamide B based on a intramolecular Heck reaction, which led to a revision of the initially proposed structure for 2 [3].Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

El análogo de las estrigolactonas GR-24 inhibe la angiogénesis in vitro e in vivo

Muchas fitohormonas han mostrado un gran potencial en la prevención y terapia contra el cáncer. Las estrigolactonas son hormonas vegetales derivadas de los caroteinoides que están implicadas en la inhibición de la ramificación de la raíz y el brote, promover la germinación de plantas parásitas e intervenir en el establecimiento de simbiosis con micorrizas arbusculares. Se ha descrito la capacidad antitumoral de diferentes análogos de estrigolactonas, entre ellos GR-24, frente a diferentes líneas celulares tumorales in vitro y en modelos xenográficos. En este estudio se ha evaluado la capacidad citotóxica y anti-angiogénica de GR-24, tanto in vitro como in vivo. In vitro, GR-24 presenta una IC50 entre 50 y 90 μM en diversas líneas celulares tumorales y endoteliales. Además, afecta a pasos clave del proceso angiogénico, como son la proliferación, diferenciación, migración y capacidad de degradación de la matriz extracelular de células endoteliales, a concentraciones menores que su IC50. En los ensayos in vivo, GR-24 muestra un gran efecto inhibidor sobre la formación de vasos sanguíneos en la membrana corioalantoidea de pollo y sobre la formación de vasos intersegmentales en embriones de Danio rerio. En conjunto, estos resultados sugieren que GR-24 puede ser un nuevo compuesto prometedor en la terapia anti-angiogénica y otras enfermedades dependientes de angiogénesis.[Our experimental work is supported by grants BIO2014-56092-R (MINECO and FEDER) and P12-CTS-1507 (Andalusian Government and FEDER) and funds from group BIO-267 (Andalusian Government). The "CIBER de Enfermedades Raras" is an initiative from the ISCIII (Spain)]. This communicaction has the support of a travel grant "Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech"