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Proteomics reveals potential non-neuronal cholinergic receptor-effectors in endothelial cells.

ABSTRACT: AIM: The non-neuronal acetylcholine system (NNAS) in endothelial cells participates in modulating endothelial function, vascular tone, angiogenesis and inflammation, thus plays a critical role in cardiovascular diseases. In this study, we used a proteomic approach to study potential downstream receptor-effectors of NNAS that were involved in regulating cellular function in endothelial cells. METHODS: Human umbilical vein endothelial cells were incubated in the presence of acetylcholine, oxotremorine, pilocarpine or nicotine at the concentration of 10 ?mol/L for 12 h, and the expressed proteins in the cells were separated and identified with two-dimensional electrophoresis (2-DE) and LC-MS. The protein spots with the largest changes were identified by LC-MS. Biowork software was used for database search of the peptide mass fingerprints. RESULTS: Over 1200 polypeptides were reproducibly detected in 2-DE with a pH range of 3-10. Acetylcholine, oxotremorine, pilocarpine and nicotine treatment caused 16, 9, 8 and 9 protein spots, respectively, expressed differentially. Four protein spots were identified as destrin, FK506 binding protein 1A (FKBP1A), macrophage migration inhibitory factor (MIF) and profilin-1. Western blotting analyses showed that treatment of the cells with cholinergic agonists significantly decreased the expression of destrin, FKBP1A and MIF, and increased the expression of profilin-1. CONCLUSION: A set of proteins differentially expressed in endothelial cells in response to cholinergic agonists may have important implications for the downstream biological effects of NNAS.

SUBMITTER: Zhang YY

PROVIDER: S-EPMC4155524 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Proteomics reveals potential non-neuronal cholinergic receptor-effectors in endothelial cells.

Zhang Yuan-yuan YY Shen Wei W Zhang Lian-cheng LC Pan Zhi-yuan ZY Long Chao-liang CL Cui Wen-yu WY Zhang Yan-fang YF Wang Hai H

Acta pharmacologica Sinica 20140804 9

<h4>Aim</h4>The non-neuronal acetylcholine system (NNAS) in endothelial cells participates in modulating endothelial function, vascular tone, angiogenesis and inflammation, thus plays a critical role in cardiovascular diseases. In this study, we used a proteomic approach to study potential downstream receptor-effectors of NNAS that were involved in regulating cellular function in endothelial cells.<h4>Methods</h4>Human umbilical vein endothelial cells were incubated in the presence of acetylchol ...[more]

PMID: 25088000

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Project description:BackgroundThe transient receptor potential ankyrin 1 (TRPA1) channel, localized to airway sensory nerves, has been proposed to mediate airway inflammation evoked by allergen and cigarette smoke (CS) in rodents, via a neurogenic mechanism. However the limited clinical evidence for the role of neurogenic inflammation in asthma or chronic obstructive pulmonary disease raises an alternative possibility that airway inflammation is promoted by non-neuronal TRPA1.Methodology/principal findingsBy using Real-Time PCR and calcium imaging, we found that cultured human airway cells, including fibroblasts, epithelial and smooth muscle cells express functional TRPA1 channels. By using immunohistochemistry, TRPA1 staining was observed in airway epithelial and smooth muscle cells in sections taken from human airways and lung, and from airways and lung of wild-type, but not TRPA1-deficient mice. In cultured human airway epithelial and smooth muscle cells and fibroblasts, acrolein and CS extract evoked IL-8 release, a response selectively reduced by TRPA1 antagonists. Capsaicin, agonist of the transient receptor potential vanilloid 1 (TRPV1), a channel co-expressed with TRPA1 by airway sensory nerves, and acrolein or CS (TRPA1 agonists), or the neuropeptide substance P (SP), which is released from sensory nerve terminals by capsaicin, acrolein or CS), produced neurogenic inflammation in mouse airways. However, only acrolein and CS, but not capsaicin or SP, released the keratinocyte chemoattractant (CXCL-1/KC, IL-8 analogue) in bronchoalveolar lavage (BAL) fluid of wild-type mice. This effect of TRPA1 agonists was attenuated by TRPA1 antagonism or in TRPA1-deficient mice, but not by pharmacological ablation of sensory nerves.ConclusionsOur results demonstrate that, although either TRPV1 or TRPA1 activation causes airway neurogenic inflammation, solely TRPA1 activation orchestrates an additional inflammatory response which is not neurogenic. This finding suggests that non-neuronal TRPA1 in the airways is functional and potentially capable of contributing to inflammatory airway diseases.

Project description:AimsCardiac ischaemia does not elicit an efficient angiogenic response. Indeed, lack of surgical revascularization upon myocardial infarction results in cardiomyocyte death, scarring, and loss of contractile function. Clinical trials aimed at inducing therapeutic revascularization through the delivery of pro-angiogenic molecules after cardiac ischaemia have invariably failed, suggesting that endothelial cells in the heart cannot mount an efficient angiogenic response. To understand why the heart is a poorly angiogenic environment, here we compare the angiogenic response of the cardiac and skeletal muscle using a lineage tracing approach to genetically label sprouting endothelial cells.Methods and resultsWe observed that overexpression of the vascular endothelial growth factor in the skeletal muscle potently stimulated angiogenesis, resulting in the formation of a massive number of new capillaries and arterioles. In contrast, response to the same dose of the same factor in the heart was blunted and consisted in a modest increase in the number of new arterioles. By using Apelin-CreER mice to genetically label sprouting endothelial cells we observed that different pro-angiogenic stimuli activated Apelin expression in both muscle types to a similar extent, however, only in the skeletal muscle, these cells were able to sprout, form elongated vascular tubes activating Notch signalling, and became incorporated into arteries. In the heart, Apelin-positive cells transiently persisted and failed to give rise to new vessels. When we implanted cancer cells in different organs, the abortive angiogenic response in the heart resulted in a reduced expansion of the tumour mass.ConclusionOur genetic lineage tracing indicates that cardiac endothelial cells activate Apelin expression in response to pro-angiogenic stimuli but, different from those of the skeletal muscle, fail to proliferate and form mature and structured vessels. The poor angiogenic potential of the heart is associated with reduced tumour angiogenesis and growth of cancer cells.

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Proteomics reveals potential non-neuronal cholinergic receptor-effectors in endothelial cells.

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Proteomics reveals potential non-neuronal cholinergic receptor-effectors in endothelial cells.

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