2015 Reduced Granulation Tissue and Wound Strength in the Absence of ±11²1 Integrin

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    Accepted Article Preview: Published ahead of advance online publication

    www.jidonline.org Reduced Granulation Tissue and Wound Strength in the

    Absence ofa11b1 IntegrinOPEN

    Jan-Niklas Schulz, Ce dric Zeltz, Ida W Srensen, MalgorzataBarczyk, Sergio Carracedo, Ralf Hallinger, Anja Niehoff,Beate Eckes, Donald Gullberg

    Cite this article as: Jan-Niklas Schulz, Ce dric Zeltz, Ida W Srensen, Malgorzata

    Barczyk, Sergio Carracedo, Ralf Hallinger, Anja Niehoff, Beate Eckes, Donald

    Gullberg, Reduced Granulation Tissue and Wound Strength in the Absence of

    a11b1 Integrin, Journal of Investigative Dermatology accepted article preview 29

    January 2015;doi: 10.1038/jid.2015.24.

    This is a PDF file of an unedited peer-reviewed manuscript that has been accepted

    for publication. NPG are providing this early version of the manuscript as a service

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    review before it is published in its final form. Please note that during the production

    process errors may be discovered which could affect the content, and all legaldisclaimers apply.

    This work is licensed under a Creative Commons Attribution-NonCommercial-

    NoDerivs 3.0 Unported License. To view a copy of this license, visit http://

    creativecommons.org/licenses/by-nc-nd/3.0/

    Received 12 May 2014; accepted 16 January 2015; Accepted article previewonline 29 January 2015

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    1

    Reduced granulation tissue and wound strength in the absence of 111integrin

    Jan-Niklas Schulz1$, Cdric Zeltz2$, Ida W. Srensen2, Malgorzata Barczyk2, Sergio Carracedo2,

    Ralf Hallinger1, Anja Niehoff3,4, Beate Eckes1, and Donald Gullberg2

    1Department of Dermatology, University of Cologne, Germany

    2 Department of Biomedicine, Centre for Cancer Biomarkers (CCBIO), Norwegian Centre ofExcellence, University of Bergen, Norway,

    3Institute of Biomechanics & Orthopedics, German Sport University, Cologne, Germany

    4Cologne Center for Musculoskeletal Biomechanics, University of Cologne, Germany

    To whom correspondence should be addressed:Donald Gullberg, Prof, PhD, Jonas Lies vei 91, N-5009 Bergen, Norway. Phone: +47-555 86332.

    Fax: +47-555 86410 E-mail:donald.gullberg@biomed.uib.no andBeate Eckes, PhD, Kerpener Str. 62, D-50937 Cologne, Germany. Phone: +49-221-478 86777. E-mail: beate.eckes@uni-koeln.de

    Short title: 11 1 integrin in wound-healing

    Keywords: dermal fibroblasts, integrin 111, integrin 21, JNK, TGF-, collagen Ireorganization, wounding

    Abbreviations: -smooth muscle actin, -SMA; Collagen integrin bridging, COLINBRI; Granulationtissue, GT; mouse embryonic fibroblasts, MEFs; TGF-receptor, TGF-R

    $J-NS and CZ contributed equally to the study.

    2015 The Society for Investigative Dermatology

    mailto:donald.gullberg@biomed.uib.nomailto:donald.gullberg@biomed.uib.no
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    Abstract

    Previous wound healing studies have failed to define a role for either 11 or 21integrin in fibroblast-

    mediated wound contraction, suggesting the involvement of another collagen receptor in this process. Our

    previous work demonstrated that the integrin subunit 11 is highly induced during wound healing both at

    the mRNA and protein level, prompting us to investigate and dissect the role of the integrin 111during

    this process. Therefore we used mice with a global ablation of either 2or 11or both integrin subunits

    and investigated the repair of excisional wounds. Analyses of wounds demonstrated that 111 deficiency

    results in reduced granulation tissue formation and impaired wound contraction, independently of the

    presence of 21.

    Our combined in vivoand in vitrodata further demonstrate that dermal fibroblasts lacking 11 1 are

    unable to efficiently convert to myofibroblasts, resulting in scar tissue with compromised tensile strength.

    Moreover, we suggest that the reduced stability of the scar is a consequence of poor collagen remodeling

    in 11-/-wounds associated with defective TGF--dependent JNK signaling.

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    Introduction

    In dermis, fibroblast interactions with the collagen network are important in maintaining skin homeostasis

    (Sorrell and Caplan, 2004)and become essentialduring repair of skin lesions (Liuet al., 2010). During

    wound healing, numerous complex cell-matrix interactions occur. Keratinocytes migrate on fibronectin in

    the provisional matrix to seal the wound towards the outside (Margadantet al., 2009). Dermal fibroblasts

    migrate into the wound, proliferate and differentiate into myofibroblasts, which produce granulation

    tissue (GT) rich in collagen I (Driskellet al., 2013; Hinz, 2007). Myofibroblasts, which express -smooth

    muscle actin (-SMA), use cytoskeleton-driven forces to contract the wound (Tomaseket al., 2002; Wipff

    et al., 2007). Finally, the contracted GT is remodeled to restore normal tissue architecture. Although the

    importance of cell-matrix interactions for wound contraction seems to be deductive, the identity of

    participating receptors in dermis has been ambiguous.

    In the skin, the collagen-binding integrins 11 and 21 are present on fibroblasts and on

    microvasculature,but only 21is expressed on basal keratinocytes (Gardneret al., 1999; Grenacheet

    al., 2007;Zweerset al., 2007). Wound healing studies in 1 1 or 21 integrin deficient mice have

    surprisingly failed to define a role for either receptor in fibroblast-mediated wound contraction (Gardner

    et al., 1999; Grenacheet al., 2007;Zweerset al., 2007), suggesting that additional collagen receptor(s)

    are involved in this process. One possible candidate is which in skin is exclusively expressed on

    fibroblasts (Vellinget al., 1999;Zhanget al., 2006).

    Early remodeling of collagen matrices mediated by fibroblasts in vitrohas been shown to be essentially

    an RGD-independent but 1 integrin-dependent process (Gullberget al., 1990). Data collected so far on

    fibroblasts have identified integrins 21 (Kleinet al., 1991;Zhanget al., 2006)and 111 (Popovaet

    al., 2007)being involved in collagen remodeling. 21 has been shown to contribute to collagen gel

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    remodeling in fibroblasts of different origin, including dermis, whereas functional studies on 111 have

    so far been limited to mouse embryonic fibroblasts (MEFs) and periodontal ligament fibroblasts (Barczyk

    et al., 2013;Popovaet al., 2007).

    In the present study, we demonstrate that repair of skin wounds is compromised in mice lacking integrin

    111, characterized by diminished wound contraction, reduced formation of GT and altered scar

    stability. Our combined in vivoand in vitrodata show that myofibroblast differentiation and collagen

    remodeling are impaired in the absence of 111. Hence, we demonstrate that efficient collagen

    remodeling requires both 111 and non-canonical TGF-1-dependent JNK signaling.

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    5

    Results

    Impaired wound healing in 11 1-deficient mice

    In previous studies we showed that expression levels of the integrin subunits 2 and 11 are elevated

    during the course of wound healing, with a peak of integrin 11 at 7 days after injury (Zweerset al.,

    2007). To dissect whether these two integrins have distinguishable functions during tissue regeneration,

    we investigated wound repair in mice that are deficient of 2 1, 11 1 or both integrins. Specifically, we

    analyzed the formation of GT and contraction of full-thickness skin wounds 7 days after lesion. Sections

    through the middle of such wounds are illustrated in Figure 1a, showing GT of C57Bl/6 wild type mice

    and their integrin-deficient littermates. As expected, the amount of GT developed in 2-null mice did not

    differ from wild type mice (Zweerset al., 2007), however significantly less GT was developed by 11-

    null mice (P=0.0317) and by double mutants (P=0.0369) (Figure 1b). As there was no difference between

    the single 11-null and the double 2/ 11-null mutant wounds, we conclude that the effect seen in the

    double mutant is attributed to the absence of 11 1 integrin. Differences in wound area were not

    reflected by the scab size, which was comparable in all 3 mutants and wild type mice (Supplementary

    figure S1a and b). However, histology revealed that the distances between wound edges - which serve as

    an indicator of wound contraction - were significantly increased in integrin 11 1-deficient wounds

    (P=0.0447; Figure 1d). The reduced wound contraction was not caused by an impaired function of the

    panniculus carnosus muscle that participates in the contraction, as it was equally contracted in all

    genotypes (Figure 1c). Since we identified lack of 11 as responsible for reduced GT formation and

    impaired wound contraction, we limited our further analysis to comparison of wounds in 11 1