Teresa Tejerina

Secretome analysis of atherosclerotic and non-atherosclerotic arteries reveals dynamic extracellular remodeling during pathogenesis☆

AIMS Early detection of cardiovascular diseases and knowledge of underlying mechanisms is essential. Tissue secretome studies resemble more closely to the in vivo situation, showing a much narrower protein concentrations dynamic range than plasma. This study was aimed to the analysis of human arterial tissue secretome and to the quantitative comparison of healthy and atherosclerotic secretome to discover proteins with key roles in atherosclerosis development. METHODS AND RESULTS Secretomes from three biological replicates of human atherosclerotic coronary arteries (APC), preatherosclerotic coronaries (PC) and mammaries (M) were analyzed by LC-MS/MS. The identified proteins were submitted to Ingenuity Pathway Analysis (IPA) tool. Label-free MS/MS based quantification was performed and validated by immunohistochemistry. 64 proteins were identified in the 3 replicates of at least one of the 3 groups and 15 secreted proteins have not been previously reported in plasma. Four proteins were significantly released in higher amounts by mammary tissue: gelsolin, vinculin, lamin A/C and phosphoglucomutase 5. CONCLUSION The study of tissue secretome reveals key proteins involved in atherosclerosis which have not been previously reported in plasma. Novel proteins are here highlighted which could be potential therapeutic targets in clinical practice. This article is part of a Special Issue entitled: Proteomics: The clinical link.

Electrophysiological effects of propafenone in untreated and propafenone‐pretreated guinea‐pig atrial and ventricular muscle fibres

1 The electrophysiological effects of propafenone (10−7 to 10−4 M) were studied on guinea‐pig isolated atrial and ventricular muscle fibres obtained from untreated animals and animals pretreated with propafenone, 3 and 10 mg kg−1, for 28 days. 2 In untreated atria propafenone produced a dose‐dependent decrease in the rate and maximum following frequency, prolonged the sinus node recovery time and reduced the maximum chronotropic responses to isoprenaline. 3 In untreated atrial and ventricular muscle fibres propafenone depressed action potential amplitude and Vmax, reduced the resting membrane potential and prolonged the action potential duration (APD) and the effective refractory period, lengthening the effective refractory period relative to APD. 4 Propafenone depressed the amplitude and Vmax and shortened the duration of the slow action potentials induced by isoprenaline and caffeine in K‐depolarized papillary muscles. 5 Pretreatment with propafenone reduced atrial rate, but did not modify the action potential characteristics compared to the values obtained in untreated atria. Further addition of propafenone produced similar but more marked changes in untreated atria. 6 In ventricular muscle fibres pretreated with 3 mg kg−1, action potential characteristics before and after further addition of propafenone were similar to those obtained in untreated fibres. However, muscles pretreated with 10 mg kg−1 exhibited a significant prolongation of the APD compared to that in untreated muscles or those pretreated with 3 mg kg−1; further addition of propafenone shortened the APD even when this parameter was of similar value to those observed in the other two series of experiments. 7 It is concluded that even though the effects of propafenone are similar to those of quinidine (class I antiarrhythmic), it also exhibited class II and class IV actions. In pretreated animals a prolongation of the APD (class III action) could also be involved in the antiarrhythmic effects of the drug.

Electrophysiological effects of platelet-activating factor (PAF-acether) in guinea-pig papillary muscles.

The effects of PAF-acether (10(-11) to 10(-7) M) were studied on the electrical and mechanical activity of guinea-pig papillary muscles. At 10(-11) M PAF-acether did not modify the amplitude and Vmax of the upstroke or the resting membrane potential. At higher concentrations PAF-acether produced a dose-dependent increase in the amplitude and Vmax of the upstroke, shortened the action potential duration and hyperpolarized the resting membrane potential. These effects were accompanied by a biphasic effect on ventricular contractile force. The shortening of the APD was inhibited in muscles pretreated with tetraethylammonium or verapamil. In papillary muscles depolarized by 27 mM K Tyrode solution PAF-acether induced slow action potentials which were blocked by verapamil. PAF-acether produced a dose-dependent increase in amplitude and Vmax of the upstroke on the slow action potentials elicited by isoproterenol, prolonged the action potential duration and hyperpolarized the resting membrane potential. These results suggest that in guinea-pig papillary muscles PAF-acether increased Ca influx via the slow inward current.

Acetylsalicylic Acid Inhibits Cell Proliferation by Involving Transforming Growth Factor-β

Background—Acetylsalicylic acid (ASA) inhibits cell proliferation. This may be mediated by transforming growth factor-&bgr; (TGF-&bgr;). TGF-&bgr; directly stops cell proliferation, restrains cells in G0, and inhibits the uptake of platelet-derived growth factor and insulin-like growth factor. These effects are identical to those observed with ASA treatment. Methods and Results—We cultured rat thoracic aorta vascular smooth muscle cells and measured cytotoxicity, cell proliferation, cell cycle, transcription of TGF-&bgr;1, and concentration of TGF-&bgr;1 in supernatant medium. ASA dose-dependently restrained cells in G0 phase with no cytotoxic effect and inhibited cell proliferation by 30.86%. Anti–TGF-&bgr;1 reversed this inhibition by 30.21%. However, ASA treatment decreased TGF-&bgr;1 transcription and had no significant effect on TGF-&bgr;1 concentration. Conclusions—TGF-&bgr; seems to play an important role in ASA-mediated inhibition of cell proliferation. Therefore, treatment with ASA prevents coronary disease not only by means of its antiplatelet properties but also by an important inhibition of plaque growth. This relationship between ASA and TGF-&bgr; explains many other effects, such as cancer chemoprevention, immunomodulation, and wound healing. The aim of this study was to demonstrate this link.

Mercury induces proliferation and reduces cell size in vascular smooth muscle cells through MAPK, oxidative stress and cyclooxygenase-2 pathways

Mercury exposure is known to increase cardiovascular risk but the underlying cellular mechanisms remain undetermined. We analyzed whether chronic exposure to HgCl2 affects vascular structure and the functional properties of vascular smooth muscle cells (VSMC) through oxidative stress/cyclooxygenase-2 dependent pathways. Mesenteric resistance arteries and aortas from Wistar rats treated with HgCl2 (first dose 4.6mgkg(-1), subsequent doses 0.07mgkg(-1)day(-1), 30days) and cultured aortic VSMC stimulated with HgCl2 (0.05-5μg/ml) were used. Treatment of rats with HgCl2 decreased wall thickness of the resistance and conductance vasculature, increased the number of SMC within the media and decreased SMC nucleus size. In VSMCs, exposure to HgCl2: 1) induced a proliferative response and a reduction in cell size; 2) increased superoxide anion production, NADPH oxidase activity, gene and/or protein levels of the NADPH oxidase subunit NOX-1, the EC- and Mn-superoxide dismutases and cyclooxygenase-2 (COX-2); 3) induced activation of ERK1/2 and p38 MAPK. Both antioxidants and COX-2 inhibitors normalized the proliferative response and the altered cell size induced by HgCl2. Blockade of ERK1/2 and p38 signaling pathways abolished the HgCl2-induced Nox1 and COX-2 expression and normalized the alterations induced by mercury in cell proliferation and size. In conclusion, long exposure of VSMC to low doses of mercury activates MAPK signaling pathways that result in activation of inflammatory proteins such as NADPH oxidase and COX-2 that in turn induce proliferation of VSMC and changes in cell size. These findings offer further evidence that mercury might be considered an environmental risk factor for cardiovascular disease.

Selected CD133+ Progenitor Cells to Promote Angiogenesis in Patients With Refractory Angina Final Results of the PROGENITOR Randomized Trial

Rationale: Refractory angina constitutes a clinical problem. Objective: The aim of this study was to assess the safety and the feasibility of transendocardial injection of CD133+ cells to foster angiogenesis in patients with refractory angina. Methods and Results: In this randomized, double-blinded, multicenter controlled trial, eligible patients were treated with granulocyte colony-stimulating factor, underwent an apheresis and electromechanical mapping, and were randomized to receive treatment with CD133+ cells or no treatment. The primary end point was the safety of transendocardial injection of CD133+ cells, as measured by the occurrence of major adverse cardiac and cerebrovascular event at 6 months. Secondary end points analyzed the efficacy. Twenty-eight patients were included (n=19 treatment; n=9 control). At 6 months, 1 patient in each group had ventricular fibrillation and 1 patient in each group died. One patient (treatment group) had a cardiac tamponade during mapping. There were no significant differences between groups with respect to efficacy parameters; however, the comparison within groups showed a significant improvement in the number of angina episodes per month (median absolute difference, −8.5 [95% confidence interval, −15.0 to −4.0]) and in angina functional class in the treatment arm but not in the control group. At 6 months, only 1 simple-photon emission computed tomography (SPECT) parameter: summed score improved significantly in the treatment group at rest and at stress (median absolute difference, −1.0 [95% confidence interval, −1.9 to −0.1]) but not in the control arm. Conclusions: Our findings support feasibility and safety of transendocardial injection of CD133+ cells in patients with refractory angina. The promising clinical results and favorable data observed in SPECT summed score may set up the basis to test the efficacy of cell therapy in a larger randomized trial.

Relaxant effects of L‐citrulline in rabbit vascular smooth muscle

1 Vascular endothelium plays a pivotal role in the control of vascular tone through the release of vasoactive factors such as EDRF (NO). 2 The aim of this study was to investigate whether the addition of exogenous L‐citrulline, the byproduct of the NO‐synthesis, could relax vascular smooth muscle. 3 L‐citrulline relaxed both endothelium‐denuded and endothelium‐intact rabbit aortic rings precontracted with noradrenaline 10−6 M (maximum relaxations induced by L‐citrulline 10−8 M were 74.1±5.2% vs 51.3±2.8% in endothelium‐denuded and endothelium‐intact arteries, respectively). 4 This relaxant effect was enhanced by zaprinast (a phosphodiesterase type 5 inhibitor) and inhibited by HS‐142‐1 (a particulate guanylate cyclase inhibitor) and by apamin (a KCa‐channel blocker). 5 L‐citrulline (10−13–10−8 M) increased cGMP levels in aortic rings (maximum value with L‐citrulline 10−8 M was 0.165±0.010 pmol cGMP mg−1 of tissue vs 0.038±0.009 pmol mg−1 of tissue in basal). 6 L‐citrulline as well as NO were released from endothelial cells in culture stimulated with ACh. The values were 6.50±0.50 μM vs 2.30±0.20 μM (stimulated with ACh and basal respectively) for L‐citrulline and 4.22±0.10 μM vs 0.87±0.26 μM (stimulated with ACh and basal respectively) for NO. 7 These results suggest that L‐citrulline could be released together with NO from endothelium and may have actions complementary to those of NO in the control of vascular smooth muscle relaxation.

Effects of calcium dobesilate on the synthesis of endothelium‐dependent relaxing factors in rabbit isolated aorta

Some cardiovascular disturbances which occur in diabetics are a consequence of alterations in vascular contractility as well as in endothelium‐dependent relaxation. Calcium dobesilate (DOBE) is a drug used in diabetic retinopathy and its mechanism of action is not yet understood. The aim of this study was to investigate the effects of DOBE on synthesis and release of endothelium‐dependent relaxing factor (EDRF) and endothelium‐dependent hyperpolarizing factor (EDHF) in rabbit isolated aorta. Endothelium‐dependent relaxation induced by acetylcholine (ACh) (10−8–10−5 M) increased in the presence of DOBE 10−5 M only when vascular endothelium was kept intact. NG‐nitro‐L‐arginine methyl ester (L‐NAME; 10−8–10−4 M progressively decreased the enhancing effect of DOBE on endothelium‐dependent relaxation whereas it was progressively increased by L‐Arg. DOBE 10−5 M increased in a non‐significant manner endothelium‐dependent relaxation induced by ACh when the arteries were incubated with both L‐NAME 10−4 M and indomethacin 10−6 M. DOBE (10−6 M and 10−5 M) was able to scavenge superoxide anion radicals generated by the hypoxanthine/xanthine oxidase reaction. These results provide evidence that DOBE is able to affect the vascular disorders associated with diabetes mellitus since it enhances the synthesis of endothelium‐dependent relaxing factors.

Biphasic effect of pioglitazone on isolated human endothelial progenitor cells: Involvement of peroxisome proliferator-activated receptor-γ and transforming growth factor-β1

Endothelial progenitor cells (EPCs) have been implicated in vascular repair and found to be functionally impaired in patients with diabetes. We evaluated the effects of the anti-diabetic drug pioglitazone on human EPC function and the involvement of PPAR-gamma and TGF-beta1. EPCs in culture were characterized at day 7 by the development of colony-forming units (CFUs) and flow cytometry assessment of differentiation marker (DiI-ac-LDL/lectin, KDR and CD31). Adhesion on fibronectin and fibrinogen in flow was analyzed as functional parameter. Treatment with pioglitazone for 72 hours increased the number of EPC-CFUs, DiI-ac-LDL(+)/lectin(+), CD31(+) and KDR(+) EPCs at 1 microM but not at 10 microM. Since pioglitazone did not significantly alter proliferation and apoptosis in cultured EPCs, the increase in EPC number was most likely attributable to augmented adhesion and differentiation. Indeed, pioglitazone increased EPC adhesion in flow at 1 microM, an effect prevented by PPAR-gamma and beta2-integrin blockade. In contrast, pioglitazone did not promote EPC adhesion at 10 microM; however, increased adhesion became evident by co-incubation with a blocking TGF-beta1 antibody. As determined by ELISA, pioglitazone induced a persistent increase in TGF-beta1 secretion only at 10 microM when a significantly elevated expression of endoglin, the accessory receptor for TGF-beta1, was also observed. Taken together, pioglitazone exerts biphasic effects on the function of isolated EPCs, causing a PPAR-gamma-dependent stimulation at 1 microM and a TGF-beta1-mediated suppression at 10 microM. These results may help to define optimal therapeutic doses of pioglitazone for improving endothelial dysfunction.

High-reproducible flow cytometric endothelial progenitor cell determination in human peripheral blood as CD34+/CD144+/CD3- lymphocyte sub-population.

Although determination of circulating endothelial progenitor cell (EPC) in peripheral blood by flow cytometry is an emerging marker for cardiovascular medicine, a common standardized protocol is still not available, due to the low numbers achieved in peripheral blood. In the present paper we describe a novel technique for EPC quantification as CD34+/CD144+/CD3- cells within the lymphocyte gate, which increases the percentages of EPC positivity described before and also offers high intra-assay reproducibility. These improvements are based on a gating strategy for big-sized lymphocytes, smooth fixation and cytometric clearance of CD3+ lymphocytes (T-cells). This last procedure is able to increase intra-assay Pearsons correlation from 0.8517 to 0.8908. Therefore, the technical setting described here offers a high-performance and clinically oriented EPC determination strategy in human peripheral blood.