Substrate regulation of vascular endothelial cell morphology and alignment

HAL Id: hal-03101118

https://hal.archives-ouvertes.fr/hal-03101118

Submitted on 7 Jan 2021

HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Substrate regulation of vascular endothelial cell morphology and alignment

A. Barakat, C. Natale, C. Leclech, J. Lafaurie-Janvore, A. Babataheri

To cite this version:

A. Barakat, C. Natale, C. Leclech, J. Lafaurie-Janvore, A. Babataheri. Substrate regulation of vascular endothelial cell morphology and alignment. Computer Methods in Biomechanics and Biomedical Engineering, Taylor & Francis, 2019, 22 (sup1), pp.S365-S366. �10.1080/10255842.2020.1714946�.

�hal-03101118�

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=gcmb20

Computer Methods in Biomechanics and Biomedical Engineering

ISSN: 1025-5842 (Print) 1476-8259 (Online) Journal homepage: https://www.tandfonline.com/loi/gcmb20

Substrate regulation of vascular endothelial cell morphology and alignment

A. I. Barakat, C. F. Natale, C. Leclech, J. Lafaurie-Janvore & A. Babataheri

To cite this article: A. I. Barakat, C. F. Natale, C. Leclech, J. Lafaurie-Janvore & A.

Babataheri (2019) Substrate regulation of vascular endothelial cell morphology and alignment, Computer Methods in Biomechanics and Biomedical Engineering, 22:sup1, S365-S366, DOI:

10.1080/10255842.2020.1714946

To link to this article: https://doi.org/10.1080/10255842.2020.1714946

© 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group

Published online: 22 May 2020.

Submit your article to this journal

Article views: 89

View related articles

View Crossmark data

Substrate regulation of vascular endothelial cell morphology and alignment

A. I. Barakat^a, C. F. Natale^a,b, C. Leclech^a, J. Lafaurie-Janvore^c and A. Babataheri^a

aLaboratoire d’Hydrodynamique, CNRS UMR7646, Ecole Polytechnique, Palaiseau, France;^bResearch Center on Biomaterials, University of Naples Federico II, Naples, Italy;

cSensome SAS, Massy, France

1. Introduction

In many cell types, function is strongly tied to shape.

This is particularly true for vascular endothelial cells (ECs) where polygonal (round) shapes have been cor- related with a pro-inflammatory phenotype that is susceptible to atherosclerosis whereas an elongated cellular morphology and alignment in the direction of blood flow correspond to an anti-inflammatory profile that remains largely protected from the disease.

Therefore, understanding how EC shape and orienta- tion are regulated is of importance.

EC shape and orientation have long been known to be regulated by the flow field to which the cells are sub- jected; however, recent

in vitro

studies have demonstrated that EC shape can also be regulated by the substrate on which the cells are cultured. For instance, significant EC elongation can be induced by culturing the cells on pla- nar patterned surfaces with selectively-defined motifs of adhesive and non-adhesive zones. Cell elongation can also be induced by plating the cells on topographic surfa- ces consisting of series of nano- or micro-scale gratings (ridges and grooves). It remains unclear, however, how ECs perceive these different types of patterned substrates and if the effects of the planar bio-adhesive and the topo- graphic substrates on ECs occur

via

similar mechanisms.

The goal of the present study is to quantitatively charac- terize EC elongation and alignment on both planar and topographic patterned surfaces of similar dimensions and to shed light onto the underlying mechanisms.

2. Methods

Planar micropatterned (mP) substrates containing alternating 5

mm-wide adhesive and non-adhesive

stripes were produced on PDMS using the deep UV light method (Azioune et al.

2010.). Topographic

micrograting (mG) substrates were fabricated by rep- lica molding of PDMS on a silicon master containing straight channels with a groove/ridge width of 5

m

m and a ridge height of 1

mm. In some experiments,

other ridge/groove dimensions and larger ridge heights (up to 7

mm)

were also investigated.

Unpatterned PDMS substrates served as controls.

Prior to cell seeding, all substrates were incubated with a 50

m

g/mL fibronectin solution in PBS for 1 hr.

In other experiments, fibronectin was adsorbed select- ively on the ridges (but not the grooves) of the topo- graphic micrograting substrates by using microcontact printing to produce a topographic surface (mG-FnR) with similar adhesive regions as the

m

P substrate.

Bovine aortic ECs (BAECs) were seeded on the differ- ent substrates, fixed and immunostained for vinculin (FAs) and actin either 2 or 24 hrs after seeding.

Quantification of FA distribution and of cell alignment and elongation was performed using Fiji software.

3. Results and discussion

On the

mG substrates, FAs were distributed uniformly

on both the ridges and grooves. In contrast, on the

mP and mG-FnR substrates vinculin concentration

exhibited sharp peaks along the edges of the adhesive zones (adhesive lines on

m

P and functionalized ridges on

mG-FnR), suggesting FA clustering in those

regions. To understand how the FA distribution affected cell morphology and orientation, cell elong- ation and alignment relative to the pattern direction were quantified. ECs on all patterned substrates were aligned in the pattern direction while cells on unpat- terned surfaces expectedly showed a random orienta- tion. Interestingly, ECs on the

mP substrates were

significantly more elongated than cells on the unpat- terned substrates as expected but surprisingly also then cells on the

m

G substrates. ECs on the

m

G-FnR substrate were even more elongated than those on the

mP substrate, suggesting that FA organization is the

primary determinant of EC elongation.

FAs organize actin stress fibers in cells; therefore, we wondered if FA clustering along pattern edges led to a particular stress fiber organization. To address

ß2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

COMPUTER METHODS IN BIOMECHANICS AND BIOMEDICAL ENGINEERING 2019, VOL. 22, NO. S1, S365–S366

https://doi.org/10.1080/10255842.2020.1714946

this issue, we used confocal microscopy to visualize the spatial distribution of actin filaments in ECs cul- tured on all the different surfaces. In ECs cultured on unpatterned substrates, stress fibers were randomly oriented with dense peripheral actin microfilament bundles, typical of ECs cultured under static (no flow) conditions (Prasain and Stevens

2009). On the m

P substrates, bundles of actin fibers at the EC basal plane were present in between adjacent fibronectin stripes, bridging FAs located at the borders of neigh- boring adhesive areas. In ECs cultured on

mG surfa-

ces, confocal z-stack imaging revealed two distinct stress fiber arrangements: stress fibers on the ridges had no clear spatial organization, whereas stress fibers in the grooves formed packed bundles oriented in the pattern direction. These bundles were associated with long and well aligned FAs detected inside the groove, suggesting that the groove surface provided direc- tional guidance for the spatial organization of stress fibers. When ECs were cultured on

mG-FnR surfaces

in which the groove was no longer accessible for adhesion, the actin network exhibited similarities to that in cells on

mP substrates, most notably suspended

thick stress fiber bundles that connected FAs located at the boundaries of neighboring adhesive ridges and thus presumably formed suspended bridges between adjacent ridges.

A key question is how FA clustering and the resulting stress fiber organization promote cellular elongation. One possibility is that the contractile stress fiber cables that run laterally over the non- adhesive stripes on the

m

P and

m

G-FnR substrates reduce the capacity of ECs to extend orthogonal to the pattern in a manner somewhat similar to the mechanism proposed by Thery et al. (2006). A second possibility relates to the dynamic nature of FAs, which can glide on surfaces due to traction forces in a treadmilling-like manner (Wolfenson et al.

2009). In

the case of the

mP and mG-FnR substrates, FAs are

separated by the non- adhesive stripes or grooves.

The resulting inhibition of FA movement acts to resist stress fiber contraction, ultimately stabilizing FA-stress fiber assembly. The validity of either or both of these potential mechanisms of cellular elong- ation remains to be investigated.

4. Conclusions

The present findings indicate that on patterned sub- strates, FA clustering regulates EC morphology through an effect on cytoskeletal organization. These results provide new insight into how substrate topog- raphy and patterning regulate EC shape and orienta- tion and promise to inform strategies of substrate engineering to target specific EC functionality.

Funding

Work supported in part by an endowment in Cardiovascular Bioengineering from the AXA Research Fund and a research grant from the Fondation Lefoulon Delalande.

References

Azioune A, Carpi N, Tseng Q, Thery M, Piel M. 2010.

Protein micropatterns: a direct printing protocol using deep UVs. Methods Cell Biol. 97:133–146.

Prasain N, Stevens T. 2009. The actin cytoskeleton in endo- thelial cell phenotypes. Microvasc Res. 77(1):53–63.

Thery M, Pepin A, Dressaire E, Chen Y, Bornens M. 2006.

Cell distribution of stress fibres in response to the geom- etry of the adhesive environment. Cell Motil Cytoskel.

63:41–55.

Wolfenson H, Henis Y, Geiger B, Bershadsky AD. 2009.

The heel and toe of the cell’s foot: a multifaceted approach for understanding the structure and dynamics of focal adhesions. Cell Motil Cytoskeleton. 66(11):

1017–1029.

KEYWORDSEndothelial cells; morphology; alignment; atherosclerosis;

patterned surfaces

barakat@ladhyx.polytechnique.fr

S366 ABSTRACT