Automated Intracellular Calcium Profiles Extraction from Endothelial Cells Using Digital Fluorescence Images

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Abstract

Endothelial cells perform a wide variety of fundamental functions for the cardiovascular system, their proliferation and migration being strongly regulated by their intracellular calcium concentration. Hence it is extremely important to carefully measure endothelial calcium signals under different stimuli. A proposal to automate the intracellular calcium profiles extraction from fluorescence image sequences is presented. Digital image processing techniques were combined with a multi-target tracking approach supported by Kalman estimation. The system was tested with image sequences from two different stimuli. The first one was a chemical stimulus, that is, ATP, which caused small movements in the cells trajectories, thereby suggesting that the bath application of the agonist does not generate significant artifacts. The second one was a mechanical stimulus delivered by a glass microelectrode, which caused major changes in cell trajectories. The importance of the tracking block is evidenced since more accurate profiles were extracted, mainly for cells closest to the stimulated area. Two important contributions of this work are the automatic relocation of the region of interest assigned to the cells and the possibility of data extraction from big image sets in efficient and expedite way. The system may adapt to different kind of cell images and may allow the extraction of other useful features.

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Most cited references 22

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Update on vascular endothelial Ca(2+) signalling: A tale of ion channels, pumps and transporters.

Roberto Romani, Francesco Moccia, Franco Tanzi (2012)

A monolayer of endothelial cells (ECs) lines the lumen of blood vessels and forms a multifunctional transducing organ that mediates a plethora of cardiovascular processes. The activation of ECs from as state of quiescence is, therefore, regarded among the early events leading to the onset and progression of potentially lethal diseases, such as hypertension, myocardial infarction, brain stroke, and tumor. Intracellular Ca(2+) signals have long been know to play a central role in the complex network of signaling pathways regulating the endothelial functions. Notably, recent work has outlined how any change in the pattern of expression of endothelial channels, transporters and pumps involved in the modulation of intracellular Ca(2+) levels may dramatically affect whole body homeostasis. Vascular ECs may react to both mechanical and chemical stimuli by generating a variety of intracellular Ca(2+) signals, ranging from brief, localized Ca(2+) pulses to prolonged Ca(2+) oscillations engulfing the whole cytoplasm. The well-defined spatiotemporal profile of the subcellular Ca(2+) signals elicited in ECs by specific extracellular inputs depends on the interaction between Ca(2+) releasing channels, which are located both on the plasma membrane and in a number of intracellular organelles, and Ca(2+) removing systems. The present article aims to summarize both the past and recent literature in the field to provide a clear-cut picture of our current knowledge on the molecular nature and the role played by the components of the Ca(2+) machinery in vascular ECs under both physiological and pathological conditions.

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Dipeptidyl Peptidase‐4 Inhibitors Attenuate Endothelial Function as Evaluated by Flow‐Mediated Vasodilatation in Type 2 Diabetic Patients

Makoto Ayaori, Naotsugu Iwakami, Harumi Uto‐Kondo … (2013)

Background Endothelial dysfunction is an independent predictor for cardiovascular events in patients with type 2 diabetes (T2DM). Glucagon like peptide‐1 (GLP‐1) reportedly exerts vasodilatory actions, and inhibitors of dipeptidyl peptidase‐4 (DPP‐4), an enzyme‐degrading GLP‐1, are widely used to treat T2DM. We therefore hypothesized that DPP‐4 inhibitors (DPP‐4Is) improve endothelial function in T2DM patients and performed 2 prospective, randomized crossover trials to compare the DPP‐4I sitagliptin and an α‐glucosidase inhibitor, voglibose (in study 1) and the DPP‐4Is sitagliptin and alogliptin (in study 2). Methods and Results In study 1, 24 men with T2DM (46±5 years) were randomized to sitagliptin or voglibose for 6 weeks without washout periods. Surprisingly, sitagliptin significantly reduced flow‐mediated vasodilatation (FMD; −51% compared with baseline, P<0.05) of the brachial artery despite improved diabetic status. In contrast, voglibose did not affect FMD. To confirm this result and determine whether it is a class effect, we conducted another trial (study 2) to compare sitagliptin and alogliptin in 42 T2DM patients (66±8 years) for 6 weeks with 4‐week washout periods. Both DPP‐4Is improved glycemic control but significantly attenuated FMD (7.2/4.3%, P<0.001, before/after sitagliptin; 7.0/4.8%, P<0.001, before/after alogliptin, respectively). Interestingly, FMD reduction was less evident in subjects who were on statins or whose LDL cholesterol levels were reduced by them, but this was not correlated with parameters including DPP‐4 activity and GLP‐1 levels or diabetic parameters. Conclusions Our 2 independent trials demonstrated that DPP‐4 inhibition attenuated endothelial function as evaluated by FMD in T2DM patients. This unexpected unfavorable effect may be a class effect of DPP‐4Is. Clinical Trial Registration URL: http://center.umin.ac.jp, Unique Identifiers: UMIN000005682 (sitagliptin versus voglibose) and UMIN000005681 (sitagliptin versus alogliptin).

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Endothelial remodelling and intracellular calcium machinery.

F Moccia, F Tanzi, L Munaron (2014)

Rather being an inert barrier between vessel lumen and surrounding tissues, vascular endothelium plays a key role in the maintenance of cardiovascular homeostasis. The de-endothelialization of blood vessels is regarded as the early event that results in the onset of severe vascular disorders, including atherosclerosis, acute myocardial infarction, brain stroke, and aortic aneurysm. Restoration of the endothelial lining may be accomplished by the activation of neighbouring endothelial cells (ECs) freed by contact inhibition and by circulating endothelial progenitor cells (EPCs). Intracellular Ca(2+) signalling is essential to promote wound healing: however, the molecular underpinnings of the Ca(2+) response to injury are yet to be fully elucidated. Similarly, the components of the Ca(2+) toolkit that drive EPC incorporation into denuded vessels are far from being fully elucidated. The present review will survey the current knowledge on the role of Ca(2+) signalling in endothelial repair and in EPC activation. We propose that endothelial regeneration might be boosted by intraluminal release of specific Ca(2+) channel agonists or by gene transfer strategies aiming to enhance the expression of the most suitable Ca(2+) channels at the wound site. In this view, connexin (Cx) channels/hemichannels and store-operated Ca(2+) entry (SOCE) stand amid the most proper routes to therapeutically induce the regrowth of denuded vessels. Cx stimulation might trigger the proliferative and migratory behaviour of ECs facing the lesion site, whereas activation of SOCE is likely to favour EPC homing to the wounded vessel.

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Author and article information

Journal

Journal ID (nlm-ta): Int J Mol Sci

Journal ID (iso-abbrev): Int J Mol Sci

Journal ID (publisher-id): ijms

Title: International Journal of Molecular Sciences

Publisher: MDPI

ISSN (Electronic): 1422-0067

Publication date (Electronic): 02 November 2018

Publication date Collection: November 2018

Volume: 19

Issue: 11

Electronic Location Identifier: 3440

Affiliations

[1 ]Electronics Department, National Institute of Astrophysics, Optics and Electronics, 72840 Puebla, Mexico

[2 ]Biomedicine School, Benemérita Universidad Autónoma de Puebla, 72410 Puebla, Mexico; ajeletvargaz7@ 123456hotmail.com

[3 ]Computer Science Department, National Institute of Astrophysics, Optics and Electronics, 72840 Puebla, Mexico; pgomez@ 123456inaoep.mx

[4 ]Department of Biology and Biotechnology “Lazzaro Spallanzani”, University of Pavia, 27100 Pavia, Italy; francesco.moccia@ 123456unipv.it

Author notes

[* ]Correspondence: marcial_st@ 123456hotmail.com (M.S.-T.); jmram@ 123456inaoep.mx (J.M.R.-C.); rberra001@ 123456hotmail.com (R.B.-R.)

Author information

Pilar Gomez-Gil https://orcid.org/0000-0003-1550-6218

Francesco Moccia https://orcid.org/0000-0003-0010-0098

Roberto Berra-Romani https://orcid.org/0000-0003-4884-0925

Article

Publisher ID: ijms-19-03440

DOI: 10.3390/ijms19113440

PMC ID: 6274978

PubMed ID: 30400174

SO-VID: 5ef5c02e-0c76-4291-8a49-792670de554c

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Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

Automated Intracellular Calcium Profiles Extraction from Endothelial Cells Using Digital Fluorescence Images

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Most cited references 22

Update on vascular endothelial Ca(2+) signalling: A tale of ion channels, pumps and transporters.

Dipeptidyl Peptidase‐4 Inhibitors Attenuate Endothelial Function as Evaluated by Flow‐Mediated Vasodilatation in Type 2 Diabetic Patients

Endothelial remodelling and intracellular calcium machinery.

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