Estudio de los mecanismos contráctiles de la musculatura lisa vascular en arterias mesentéricas humanas y su modificación por el envejecimiento : papel del retículo sarcoplásmico

Se ha realizado un estudio de los procesos contráctiles de la musculatura lisa vascular en arterias humanas, comprobando las posibles implicaciones del retículo sarcoplásmico en el proceso contráctil y las diferencias que pudieran existir debidas al envejecimiento. Para ello se han utilizado arterias mesentéricas humanas de pacientes de diferentes edades, realizando el estudio con cuatro técnicas distintas: miografía vascular, para la medición de los procesos contráctiles arteriales, microscopía confocal, para la medición de los movimientos intracelulares de calcio que inician y mantienen la contracción arterial por medio de oscilaciones de calcio, microscopía electrónica, para la medición de la cantidad de retículo sarcoplásmico de las células musculares lisas vasculares y comprobar la posibilidad de rellenado del mismo con calcio extracelular, y western blotting, para la medición de la expresión de proteínas involucradas en el mecanismo de sensibilización al calcio que forma parte del proceso contráctil. A su vez se han utilizado diversos vasodilatadores (nifedipina, bloqueante de los canales de calcio activados por voltaje tipo L; KBR, inhibidor del intercambiador sodio calcio en modo reverso; SKF, bloqueante de los canales activados por receptor y almacenamiento; HA-1077, inhibidor de la encima Rho kinasa; 2-APB, bloqueante de los canales de IP3; CPA, inhibidor de la bomba de entrada de calcio del retículo sarcoplásmico SERCA) para la caracterización de los mecanismos a través de los cuales el calcio produce su efecto contráctil. Pudimos observar que el proceso de contracción en arterias humanas es dependiente de calcio extracelular y de calcio procedente del retículo sarcoplásmico, algunos de los mecanismos contráctiles involucrados se ven modificados por el envejecimiento. Los movimientos intracelulares de calcio que forman parte del proceso contráctil se presentan como oscilaciones sincrónicas. El proceso de contracción puede verse afectado por la sensibilización al calcio, pudiendo ser dependiente de la edad, mientras que en la relajación el proceso de sensibilización tiene un papel importante. [ABSTRACTS]A study of the contractile processes in smooth muscle cells from human arteries has been done, assessing the possible implication of sarcoplasmic reticulum in such process and the possible differences along age. Human mesenteric arteries from different aged patients and four different techniques were used: vascular myography, for the measuring of artery contractile processes, confocal microscopy, for measuring the calcium intracellular movements that initiate and maintains the arterial contraction with calcium oscillations, electron microscopy, for measuring the amount of sarcoplasmic reticulum in vascular smooth muscle cells and to prove the possible refilling of the sarcoplasmic reticulum with extracellular calcium, and western blotting, for measuring the expression levels of the proteins involved in the calcium sensitization pathway which is part of the contractile process. Also we have used several vasodilators (nifedipine, L-type voltage gated calcium channel blocker; KBR, inhibitor of the sodium calcium exchanger reversal mode; SKF, store and receptor operated calcium channels blocker; HA-1077, Rho kinase inhibitor; 2-APB, IP3 channel blocker; CPA, sarcoplasmic reticulum calcium ATPase SERCA blocker) for the characterization of the mechanisms through which calcium produces contraction. We observed that the human arteries contraction process is dependent on extracellular and sarcoplasmic reticulum calcium, and some of the contractile mechanisms involved are modified by ageing. The calcium intracellular movements that conforms the contractile process are synchronised oscillations. The contraction process can be affected by calcium sensitization which could be age dependent, while in the relaxing process, sensitization has a important role

Vascular Dysfunction in a Transgenic Model of Alzheimer's Disease: Effects of CB1R and CB2R Cannabinoid Agonists

There is evidence of altered vascular function, including cerebrovascular, in Alzheimer's disease (AD) and transgenic models of the disease. Indeed vasoconstrictor responses are increased, while vasodilation is reduced in both conditions. β-Amyloid (Aβ) appears to be responsible, at least in part, of alterations in vascular function. Cannabinoids, neuroprotective and anti-inflammatory agents, induce vasodilation both in vivo and in vitro. We have demonstrated a beneficial effect of cannabinoids in models of AD by preventing glial activation. In this work we have studied the effects of these compounds on vessel density in amyloid precursor protein (APP) transgenic mice, line 2576, and on altered vascular responses in aortae isolated ring. First we showed increased collagen IV positive vessels in AD brain compared to control subjects, with a similar increase in TgAPP mice, which was normalized by prolonged oral treatment with the CB1/CB2 mixed agonist WIN 55,212-2 (WIN) and the CB2 selective agonist JWH-133 (JWH). In Tg APP mice the vasoconstriction induced by phenylephrine and the thromboxane agonist U46619 was significantly increased, and no change in the vasodilation to acetylcholine (ACh) was observed. Tg APP displayed decreased vasodilation to both cannabinoid agonists, which were able to prevent decreased ACh relaxation in the presence of Aβ. In summary, we have confirmed and extended the existence of altered vascular responses in Tg APP mice. Moreover, our results suggest that treatment with cannabinoids may ameliorate the vascular responses in AD-type pathology.This work was supported by the Council of Madrid (S- BIO/0170/2006 and P2010/BMD-2349 to MLC) and by Instituto de Salud Carlos III/FISS (PI12/00590 to TT). AM received a fellowship from the Spanish Ministry of Education and Science and JN-D from FISS. Dr. R. Martínez-Murillo is acknowledged for preliminary EM experiments.Peer reviewedPeer Reviewe

Role of TGF-β1 and MAP Kinases in the Antiproliferative Effect of Aspirin in Human Vascular Smooth Muscle Cells

We aimed to test the antiproliferative effect of acetylsalicylic acid (ASA) on vascular smooth muscle cells (VSMC) from bypass surgery patients and the role of transforming growth factor beta 1 (TGF-beta1).VSMC were isolated from remaining internal mammary artery from patients who underwent bypass surgery. Cell proliferation and DNA fragmentation were assessed by ELISA. Protein expression was assessed by Western blot. ASA inhibited BrdU incorporation at 2 mM. Anti-TGF-beta1 was able to reverse this effect. ASA (2 mM) induced TGF-beta1 secretion; however it was unable to induce Smad activation. ASA increased p38(MAPK) phosphorylation in a TGF-beta1-independent manner. Anti-CD105 (endoglin) was unable to reverse the antiproliferative effect of ASA. Pre-surgical serum levels of TGF-beta1 in patients who took at antiplatelet doses ASA were assessed by ELISA and remained unchanged.In vitro antiproliferative effects of aspirin (at antiinflammatory concentration) on human VSMC obtained from bypass patients are mediated by TGF-beta1 and p38(MAPK). Pre-surgical serum levels of TGF- beta1 from bypass patients who took aspirin at antiplatelet doses did not change

Age-dependent defective TGF-beta1 signaling in patients undergoing coronary artery bypass grafting

Background: Transforming growth factor beta (TGF-beta 1) is a pleiotropic cytokine, which is deregulated in atherosclerosis; however the role of age in this process is unknown. We aimed to assess whether TGF-beta 1 signaling is affected by age. Methods: Vascular smooth muscle cells (VSMC) were obtained from patients undergoing abdominal surgery. Levels of TGF-beta 1 were measured by ELISA in sera from 169 patients undergoing coronary artery bypass grafting (CABG). The p27 expression was determined by Western blot from internal mammary arteries (IMA) obtained from CABG patients (n = 13). In VSMC from these patients undergoing abdominal surgery, secretion of TGF-beta 1 was determined by ELISA of cell-conditioned media. Results: In VSMC from aged patients we observed a lower TGF-beta 1 secretion, measured as TGF-beta 1 concentration in cell conditioned medium (p < 0.001). This effect was correlated to an age-dependent decrease of p27 expression in IMA from aged CABG patients. In a similar manner, there was an age-dependent decrease of serum TGF-beta 1 levels in CABG patients (p = 0.0195). Conclusions: VSMC from aged patients showed a higher degree of cellular senescence and it was associated to a lower TGF-beta 1 secretion and signaling.S

Evaluación de etilenglicol como crioprotector en la crioconservación de semen de bagre blanco (Sorubim cuspicaudus, Pimelodidae)

Se evaluó el semen crioconservado de Sorubim cuspicaudus utilizando etilenglicol (ETG) a tres niveles de inclusión (5, 10, 15 %). Machos (n = 13) en fase de espermiación y hembras (n = 6) en maduración final se indujeron con 0,4 ml de Ovaprim®/Kg, después de 12 a 14 horas post-inducción se colectó el semen en viales Eppendorf de 2 ml de capacidad. Las diferentes soluciones crioprotectoras se prepararon con glucosa 6 % (p/v), leche en polvo descremada 5 % (pv) y agua destilada. El semen fue diluido en proporción 1:3 (semen:diluyente), empacado en macrotubos de 2,5 ml y congelado en vapores de nitrógeno líquido (NL) durante 30 minutos y luego almacenados en termos criogénicos sumergidos directamente en NL (-196 °C). El semen crioconservado fue descongelado en baño serológico a 35 °C durante 90 segundos. La movilidad total, progresividad y velocidad espermática del semen fresco y descongelado se analizó con el software Sperm Class Analizer SCA® (Microptic SL, España). La fertilidad y eclosión se evaluó con 1,0-1,5 g de ovocitos en incubadoras experimentales de flujo ascendente de dos litros de capacidad. Se utilizó un diseño completamente aleatorizado. El semen fresco registró tasa de eclosión de 51,8±21 %, sin observarse diferencia significativa con la obtenida con el semen crioconservado con ETG 5 % (38,6 ± 13,9 %) (p 0,05); mientras que ETG 15 % (9,6 ± 2,9 %) reportó la menor eclosión (p 0,05). Los resultados sugieren que la solución crioprotectora compuesta por ETG 5 %, glucosa 6 % y leche en polvo 5 % es una alternativa viable para la crioconservación de semen de Sorubim cuspicaudus con fecundaciones similares al usar semen fresco.The catfish Sorubim cuspicaudus cryopreservation semen was evaluated using three levels (5, 10, 15 %) of ethylene glycol (ETG). Males (n = 13) undergoing spermiation and in final maturation females (n = 6) were induced with 0.4 ml Ovaprim®/Kg, after 12 and 14 post-induction the semen was collected in 2 ml Eppendorf vials. The different cryoprotectants solutions were prepared with glucose 6 % (w/v) skimmed milk powder 5 % (w/v) and distilled water. The semen was diluted in ratio 1:3 (semen:extender), packed in macrotubes of 2.5 ml and frozen in liquid nitrogen (NL) vapor for 30 minutes, then the macrotubes were stored in cryogenic tanks submerged directly in NL (-196 °C). The sperm were thawed in serological bath to 35 °C for 90 seconds. The total motility, total progressivity and velocities in fresh and thawed semen were analyzed with the Sperm Class Analyzer software SCA® (Microptic SL, Spain). Fertility and hatching rates were assessed with 1.0-1.5 g of oocytes in experimental up flow incubators two liters, a completely randomized design was used. The hatching rate of fresh semen was 51,8±21 %, with no significant differences with semen cryopreserved with ETG 5 % (38.6 ± 13.9 %) (p 0,05), while ETG 15 % (9.6 ± 2.9 %), recorded the lower hatching rate (p 0.05). The results suggest that the cryoprotectant solution composed of ETG 5 %, glucose 6 % and powdered milk 5 % is a viable alternative for semen cryopreservation of the catfish Sorubim cuspicaudu

Decreased pre-surgical CD34+/CD144+ cell number in patients undergoing coronary artery bypass grafting compared to coronary artery disease-free valvular patients

<p>Abstract</p> <p>Background</p> <p>Cardiovascular disease has been linked to endothelial progenitor cell (EPC) depletion and functional impairment in atherosclerosis and aortic stenosis. EPCs may play a pivotal role in vascular grafting. However, the EPC depletion in coronary artery bypass grafting (CABG) patients has not been compared to coronary artery disease-free valvular replacement patients with aortic stenosis.</p> <p>Methods</p> <p>We aimed to assess the basal number of CD34⁺/KDR⁺and CD34⁺/CD144⁺cells in CABG patients, compared to aortic stenosis valvular replacement patients. 100 patients (51 CABG and 49 valvular surgery ones) were included in the present study. All CABG or valvular patients had angiographic demonstration of the presence or the absence of coronary artery disease, respectively. Numbers of CD34⁺/KDR⁺and CD34⁺/CD144⁺were assessed by flow cytometry of pre-surgical blood samples.</p> <p>Results</p> <p>We found a lower number of CD34⁺/CD144⁺cells in CABG patients compared to valvular patients (0.21 ± 0.03% vs. 0.47 ± 0.08%), and this difference remained statistically significant after the <it>P </it>was adjusted for multiple comparisons (<it>P </it>= 0.01428). Both groups had more EPCs than healthy controls.</p> <p>Conclusions</p> <p>Pre-surgical CD34⁺/CD144⁺numbers are decreased in CABG patients, compared to valvular patients with absence of coronary disease.</p

Pharmacological basis and clinical evidence of dabigatran therapy

Dabigatran is an emerging oral anticoagulant which is a direct inhibitor of thrombin activity. It has been approved in the European Union and the United States of America for the prevention of thrombosis after major orthopedic surgery. It has also been approved by the American Food and Drug Administration and the European Medicines Agency for the prevention of stroke in chronic atrial fibrillation. Dabigatran provides a stable anticoagulation effect without any need to perform periodical laboratory controls. Of note, there is a growing amount of clinical evidence which shows its safety and efficacy. For these reasons, dabigatran may suppose a revolution in oral anticoagulation. However, two important limitations remain. First, it is contraindicated in patients with end-stage renal disease. Second, there is no evidence of the prevention of thrombosis in mechanical heart valves