progenitor Cells

Accepted Manuscript + Toll-like receptor-4 Signaling Improved the Migration of Sca-1 stem/progenitor Cells Ying Huang, Chunya Zhang, Jinghua Shen, Xiaogang Zhang, Jianqing Du, Daifu Zhang PII:

S0890-5096(16)31397-8

DOI:

10.1016/j.avsg.2017.07.022

Reference:

AVSG 3514

To appear in:

Annals of Vascular Surgery

Received Date: 26 December 2016 Revised Date:

0890-5096 0890-5096

Accepted Date: 18 July 2017

Please cite this article as: Huang Y, Zhang C, Shen J, Zhang X, Du J, Zhang D, Toll-like receptor-4 + Signaling Improved the Migration of Sca-1 stem/progenitor Cells, Annals of Vascular Surgery (2017), doi: 10.1016/j.avsg.2017.07.022. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof 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 legal disclaimers that apply to the journal pertain.

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Toll-like receptor-4 Signaling Improved the Migration of Sca-1+stem/progenitor

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Cells

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Ying Huang1, Chunya Zhang1, Jinghua Shen, Xiaogang Zhang, Jianqing Du, Daifu Zhang*

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Dept of Cardiology, Pudong New Area People' Hospital, Shanghai, 200120, China

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*Address correspondence to:

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Daifu Zhang, MD

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Dept of Cardiology, Pudong New Area People' Hospital, Shanghai, 200120, China

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Phone: +86(021) 66345628

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equal contribution to this paper

E-mail: [email protected]

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There is no conflict of Interest.

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Abbreviation:

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TLR, toll-like receptor; SPC, stem/progenitor cell; oxLDL, oxidized low density

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lipoprotein; MMP, Matrix metalloproteinase; PRR, pattern recognition receptor;

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NF-kB, nuclear factor kB; IRF, IFN-regulatory factor; PMB, polymyxin B.

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Abstract Atherosclerosis is considered as a chronic inflammatory process, during which

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macrophages and smooth muscle cells migrate into the vascular wall intima to initiate

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atherogenesis. Stem/progenitor cells (SPCs) have been demonstrated as a new source

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of monocytes/macrophages and smooth muscle cells (SMCs). However, the regulation

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of SPCs migration into atherosclerotic initiation sites to produce macrophages and

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SMCs in-situis not fully elucidated. As toll-like receptor (TLRs) signaling plays

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essential role in migration of immunocytes, we wanted to explore the expression and

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function of TLRs in SPCs in detail. For this we isolated Sca-1+ bone marrow cells as

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SPCs, and TLRs were detected with quantitative RT-PCR and Western blotting. Our

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data show that multiple TLRs expressed on SPCs, with TLR4 as the most prominent

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one, and activation of TLR4 in SPCs is required for migration as assessed by

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conventional wound healing and transwell assays. Knockdown of TLR4 expression

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abrogates the migration of SPCs significantly, and re-expression restores it, further

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confirming functions of TLR4 in regulating SPCs biological behavior.

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In summary, our study shows the essential function of TLR4 signaling in SPC

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migration, strongly suggesting of its role in atherosclerotic lesion formation. Our data

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provide new insight into the biological regulators in SPC migration, with potential

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consequences for therapeutics of atherosclerosis.

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Key words: toll-like receptor 4 (TLR 4); stem/progenitor cell (SPC); migration

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Introduction Atherosclerosis has been considered a chronic inflammatory process complicated

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by multiple genetic and non-genetic factors [1-5]. The atherosclerotic lesion is

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characterized by cellular infiltration of immunocytes, such as macrophages, smooth

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muscle cells (SMCs) and accumulation of non-cellular substances, such as lipids, in

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the artery wall. The process is enhanced with deposition of extracellular matrix

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composed mostly of collagens to form a fibrous cap[6].

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It has been assumed that oxidized low density lipoprotein (oxLDL), as well as

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turbulence of blood flow activates local endothelial cells, which express diverse

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adhesion molecules and chemokines, leading to transmigration of monocytes into the

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vessel wall intima. Monocytes in the intima, upon local chemokine-cytokine

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stimulation, differentiate into macrophages, which take up oxLDL and eventually

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become foam cells. Meanwhile, cytokines from activated endothelial cells lead to

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SMC migration into the intima, and proliferation to produce a variety of extracellular

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matrix components [2-4]. However, increasing evidence shows that stem/progenitor

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cells (SPCs) represent an alternative source of monocytes/macrophages and SMCs

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[7-9] to participate in the pathophysiology of atherosclerosis.

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Studies have demonstrated identified both bone marrow–derived SPCs and local

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SPCs are involved in atherosclerotic lesions, respectively, and SPCs from both

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sources are necessary for atherosclerosis to produce macrophages and SMCs [9, 10].

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Consequently, the regulators of SPC migration are of intensive interests also as

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potential sources of new therapeutics.

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The innate immune system is responsible for constant scanning and early detection

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pathogen-associated molecular patterns from microbes via a number of pattern

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recognition receptors (PRRs) [11-14]. Recent evidence, however, indicates that PRRs

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recognize damage-associated molecular patterns from injured tissue and play essential

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roles in diverse biological events, such as inflammation, tissue repair, and

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development [15-18]. Toll-like receptors (TLRs) comprise a major group of such

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PRRs. To date, 13 TLRs have been identified in mammalian cells; 10 of which (TLR1

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to TLR10) are expressed inhuman cells. These TLRs are located either on the cell

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surface or in intracellular compartments and detect diverse, distinct signals [19-21].

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Upon binding of respective ligands, TLR interacts with the TIR domain-containing

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adapters to initiate NF-kB signaling cascade and/or IFN-regulatory factor (IRF)

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signaling cascade. This leads to the production of proinflammatory cytokines and type

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I interferons, thus contributing to [4, 22, 23]. Expression of TLRs has been reported

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on immunocytes, such as macrophages and dendritic cells during atherogenic plaque

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generation.

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of

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However, the expression and function of TLRs in SPCs remain to be determined.

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In this study we report the expression pattern of different TLRs in SPCs, and explore

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the TLRs in regulating the biological behavior of SPCs. Our data show that TLR4 is

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particularly expressed in SPCs, and blocking of TLR4 signaling decrease such

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migration capabilities, suggesting of potential therapeutic effect in atherosclerosis.

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Materials and Methods 5

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Lentiviral vectors construction pLVX-shRNA-zsGreen was used to construct the recombinant lentivirus

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containing TLR4 or control shRNA sequences from Invivogen (San Diego,

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USA).TLR4 was cloned into pLVX-IRES-ZsGreen vector to construct the

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recombinant lentivirus. Recombinant lentiviruses were produced by transfecting

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HEK293T cells (ATCC, USA) with pMD.2G, psPAX2 (Takara, Japan). An MOI

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(Multiplicity of Infection) of 10 was used to inoculate SPCs, and the efficiency was

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checked 48 hours later with fluorescence microscopy.

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Isolation of Sca-1+ stem/progenitor cells

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Sca-1+SPCs were isolated from bone marrow of mice by fluorescence cell sorter

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assay as described previously [10]. In brief, bone marrow was isolated from the

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femurs and tibias of mice 6-12 weeks of age. The bone marrow was flushed out and

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subjected a 70µm nylon mesh cell strainer, and was resuspended in DMEM (Gibco

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BRL Co.Ltd.,USA) supplemented with 2%Fetal Calf Serum (FCS, Gibco BRL

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Co.Ltd.,USA). After red blood cells lysis, the bone marrow mononuclear cells were

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washed three times, and resuspended in DMEM with 5%Bovine Serum Albumin

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(BSA, Gibco BRL Co.Ltd.,USA) at 1x106cells/100µl. Next, the single cells were

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stained with control FITC-IgG(Cell Signal Technology,USA)or FITC-Sca-1 antibody

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(Cell Signal Technology ,USA), followed by fluorescence cell sorting assay using

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flow cytometry (Becton Dickinson, Heidelberg, Germany). Sca-1+ cells were

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collected in DMEM with 5% BSA and kept on ice for other experiments.

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Stem/progenitor cell migration assay

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ACCEPTED MANUSCRIPT Transwell assay was used for the cell migration assay as previously described

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[24]. Briefly, Sca-1+SPCs were trypsinization with 0.25% trypsin and 0.02% EDTA in

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PBS, counted and suspended at a density of 1×106cells/ml in 1% BSA DMEM. The

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underside of the inserts was pre-coated with 20 g/ml laminin1 (Sigma St. Louis,

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USA), or pre-grown with monolayer of endothelial cells (The cells were purchased

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from ATCC and grown in DMEM supplemented with 15% FBS).1.0 × 105cells in

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300 l serum-free medium were added into each inserts in a 24-well tissue culture

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plate and 500 l of DMEM medium containing 1% Fetal Bovine Serum (FBS, Gibco

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BRL Co.Ltd., USA),and SDF-1 (100ng/ml, Sigma St. Louis, USA), were added to the

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lower chamber. The cells in the Transwell plates were incubated at 37°C for24 h. The

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cells that had migrated into the bottom well with the cells on the lower surface were

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quantified. Migration activity was displayed as the percentage (%) of migrated cells to

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total cells. All experiments were performed in three independent experiments in

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triplicate.

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For the wound-healing assay, cells were grown in 24-well plate, and with or

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without pretreated with PMB (20ug/ml, Sigma St. Louis, USA), the cell monolayers

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were incubated with lipopolysaccharide (LPS, Sigma St. Louis, USA), for 4 hours,

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then were scraped by the sterile 26G needle to produce a wound to confluent cell layer.

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After washes with PBS, the wells were added new medium. After overnight culture at

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37°C, the cells were fixed and images were taken.

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Quantitative real-time PCR

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Total RNA was prepared from SPC cells and was reverse-transcribed using 7

ACCEPTED MANUSCRIPT qRT-PCR kit (Invitrogen, San Diego, Ca) Quantitative real-time PCR was carried out

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on an ABI Prism 7900HT (Applied Biosystems, USA). The program conditions were

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as follows:2-minute incubation at 50°C, then 95°C for 10 minutes, and followed with

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95°C for15 seconds and 60°C for 60 seconds for 40 cycles. We used GAPDN as an

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internal control to normalize for differences in the amount of total RNA in each

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sample. The primers used in this study were purchased from Invitrogen.

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Western blotting

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Cells were collected and lysed with a lysis buffer containing 1% NP-40. After

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brief vortexing and rotation, the cell lysates were separated with SDS-PAGE and

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transferred to nitrocellulose membranes (Amersham Biosciences, Piscataway,

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NJ).After 30 min blocking with blocking solution (50 mM Tri-HCl, 150 Mm NaCl,

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5% (w/v) non-fat dry milk and 0.1% Tween-20), the membrane was incubated with

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primary antibody, and then with HRP-conjugated secondary antibody(Cell Signal

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Technology, USA). After washes, the protein bands were visualized with ECL plus

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immune-blotting detection reagents (Pierce Biotechnology, Rockford, IL).

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Immunofluorescent staining

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Cells grown on cover slips were fixed in 4% freshly made paraformaldehyde for

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15 minutes, and were incubated with FITC-TLR4 antibody (Cell Signal Technology,

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USA) for 1 hour at room temperature. Images were taken with Nikon C-HGFI

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fluorescent microscope (Nikon, Japan).

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Statistics

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The 2-tailed Student’st-test or one-way ANOVA was used for statistical analysis in this research. When P＜0.05, the difference was defined as statistically significant.

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Results

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Expression of TLRs on SPC

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As reported previously, we used Sca-1+ bone marrow cells as SPCs, which are

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shown to have characteristics of progenitor cells [9]. In order to study the expression

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of TLRs on SPC, we extracted total RNA from SPCs, and used quantitative RT-PCR

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to detect the expression level of TLR1 to 9, which are well-studied and have

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well-defined ligands. Our results showed that TLR2-4 and 7-9 were expressed on

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SPCs, with prominence of TLR4 (Figure 1A). This suggests that TLR4 is potentially

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more important than the other TLRs. Accordingly we mainly focused to study TLR4

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influence on SPCs.

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Next we explored the respective protein levels using western blotting, and our

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data showed that TLR4 expressed in CD34+-BM, Sca-1+-BM, SMC, and macrophages

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was roughly similar, while substantially lower on endothelial cells (Figure 1B). These

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results unequivocally demonstrated that TLR4 is expressed on both terminally

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differentiated cells and progenitor cells such as SPCs.

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Activation of TLR4 enhances SPCs migration

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Since TLR4 was highly expressed on SPCs, so we assumed TLR4 signaling is

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involved in SPCs migration, which is essential for SPC recruitment to atherosclerotic

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lesion. Wound healing and transwell assays are two most common methods to assess

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cellular migration, so we used both to evaluate the SPCs migration upon TLR4

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activation. Lipopolysaccharide (LPS) is a well-defined and specific TLR4 ligand, so we

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stimulated SPCs with different concentrations of LPS. We found 200ng/ml to be the

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optimal concentration for SPCs migration (Figure 2A) in our assay. As shown in

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Figures 2B and C, LPS stimulation dramatically increases the migration of SPCs. The

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effect was proven specific, as abrogation of LPS binding to TLR4 with polymyxin B

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(PMB), the migration of SPCs was reversed.

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Deficiency of TLR4 reduces migration of SPCs

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Although we observed the effects of TLR4 on migration of SPCs, it could be

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other unknown mechanism of LPS on SPCs. So next we used specific shRNA to

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knockdown the TLR4 expression and to verify the role of TLR4.

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As the efficacy of transfection of SPCs with common reagents such as

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lipofectamine is very low, we produced recombinant lentivirus containing specific

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shRNAor TLR4 sequence for knockdown and over-expression, respectively. The

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efficacy of transfection was confirmed by ZsGreen expressed by the lentivirus in

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fluorescence microscopy (data not shown). The knockdown and over-expression of

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TLR4 was confirmed using immune-blotting. The results showed that knockdown of

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TLR4 expression was successful (Figure 3A and B).

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Next we used SPCs, TLR4 knockdown SPCs and TLR4 rescued SPCs to perform

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migration experiment. The results showed that the migration of SPCs with TLR4

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knockdown was greatly decreased (＜50%, Figure 3C and D). When the knockdown 10

ACCEPTED MANUSCRIPT of TLR4 was rescued by TLR4-lentivirus, the migration of SPCs was restored (Figure

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3C and D), confirming the specific effects of TLR4 on SPCs migration. Taken

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together, our data revealed that the TLR4 are required for SPCs migration and thus

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most likely are important in atherosclerosis.

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Discussion

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Increasingly amount of studies have demonstrated that the immunological

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system plays a paramount role in the pathogenesis of atherosclerosis [1, 3, 5, 25].

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Both innate and adaptive immunity effects are represented on atherosclerotic lesions.

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This includes involvement of cells such as macrophages, dendritic cells, B and T

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cells.

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SMCs are another major cell type in atherosclerotic plaques and appear to play

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key role in plaque formation and progression. Mounting evidence indicates that

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hematopoietic SPCs are an alternative source of both macrophages and SMCs, which

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are present in atherosclerotic lesions. For both cell types, the migration of SPCs is

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central for their recruitment to the atherosclerotic plaque. Earlier studies have

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suggested that some molecules are of pivotal importance in the process, such as

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Matrix metalloproteinases 8 (MMP8) and NEDD-9. As more attention focused on the

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mechanism of SPCs in atherogenesis, more regulators of SPC migration are most

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certainly going to be discovered.

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TLR is an evolutionarily conserved family of receptors, while PRR being the

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most studied and has been shown to have diverse functions beyond those in innate

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ACCEPTED MANUSCRIPT immunity accordingly [26]. Besides immunocytes, such as macrophages and dendritic

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cells, TLRs are expressed on multiple cell types, such as endothelial and epithelial

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cells [3, 13]. Recently, it has been reported that TLRs are expressed on stem cells,

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including on embryonic and mesenchymal stem cells [27, 28].

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Here our data demonstrated that different TLRs are expressed in SPCs, with

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TLR4 as the most prominent one. TLR4 is known to use both MyD88-dependent and

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–independent cascades leading to NF- B, IRF5, and mitogen-associated protein

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kinase (MAPK) signaling. This may lead to multiple biological events, such as

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migration, proliferation and differentiation[29]. During atherosclerosis, SPCs have to

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migrate from bone marrow or vascular wall to the atherosclerotic lesion. TLR4

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activation leads to production of a variety of chemokines, including monocyte

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chemotactic protein-1 (MCP-1), and Macrophage Inflammatory Protein-1, which are

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essential for diverse cell migration events. It has been reported that MMP8 was

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important during SPCs migrating into atheromas [10], and TLR4 signaling did result

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in production of MMP8, suggesting the possible pathway.

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In summary, our study shows that TLR4is expressed on SPCs, and the essential

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function of its signaling is for SPC migration, thus contributing to atherosclerotic

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lesion formation. Our data provide new insight into the biological regulators and

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mechanisms involved in the regulation of SPC migration, and may potentially assist

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in finding new therapeutics for atherosclerosis.

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Figure Legends

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Fig. 1 TLRs expression in SPCs. (A) Total RNA was prepared from SPCs and was

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reverse-transcribed using qRT-PCR kit. Different types of TLR were detected by real

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time PCR and * stands for p<0.05. The relative expression of TLR4 was significantly

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higher than others as the next target protein. (B) CD34+-BM, Sca-1+-BM, SMC,

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macrophages and endothelial cells were collected to determine the expression of

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TLR4 using western blot analysis. (C) Sca-1+-BM, macrophages and HEK293 cells

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were grown on cover slips, stained with FITC-TLR4 and subjected to fluorescent

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microscopy.

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Fig. 2 Activation of TLR4 enhances SPCs migration. (A) SPCs treated with LPS at

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different concentrations for 4 hours were used for the transwell assay and then

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transmigrated cells were counted. The SPCs treated with LPS at 200ug/ml migrated

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significantly more suitable for inducing TLR4. (B) Three SPCs were assayed using

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transwell, including SPC control, SPC treated with LPS (200ug/ml) and SPC treated

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both LPS (200ug/ml) and PMB. The cells in the transwell plates were incubated at

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37°C for 12h. Transmigrated cells were counted. (C) Wound-healing assay. Treated

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cells as indicated scraped, and after overnight culture at 37°C, the cells were fixed and

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images were taken.

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Fig. 3 Deficiency of TLR4 reduces migration of SPCs. SPCs were inoculated with

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lentivirus containing specific shRNA sequence against TLR4 (for knockdown,

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MOI=20) (A) or lentivirus containing TLR4 sequence (for over-expression, MOI=20)

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immunoblotted using TLR4 antibody. A representative image of immune-blotting

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from 3 independent experiments. (C) SPCs with TLR4 knockdown and re-expression

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subjected to transwell assay to assess the migration abilities. Transmigrated cells were

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quantified. The data is shown as Mean ±SD (n=3). (D) SPCs with TLR4 knockdown

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and re-expression subjected to wound-healing assay. Treated cells as indicated

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scraped, and after overnight culture at 37°C, the cells were fixed and images were

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taken.

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