Bone marrow-derived mesenchymal stem cells promoted cutaneous

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Bone marrow-derived mesenchymal stem cells promoted cutaneous wound healing by regulating keratinocyte migration via the #2-adrenergic receptor signaling Jiahui Huo, Sujing Sun, Zhijun Geng, Wei Sheng, Runkai Chen, Kui Ma, Xiaoyan Sun, and Xiaobing Fu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b01138 • Publication Date (Web): 14 May 2018 Downloaded from http://pubs.acs.org on May 15, 2018

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Molecular Pharmaceutics

Bone marrow-derived mesenchymal stem cells promoted cutaneous wound healing by regulating keratinocyte migration via the β2-adrenergic receptor signaling Jiahui Huo ‡ Affiliations: 1 Tianjin Medical University, No. 22, Qixiangtai Road, Heping District, Tianjin 300070, P.R.China 2 Key Research Laboratory of Tissue Repair and Regeneration of PLA, and Beijing Key Research Laboratory of Skin Injury, Repair and Regeneration, First Affiliated Hospital to the Chinese PLA General Hospital, 51 Fucheng Road, Beijing 100048, P.R.China E-mail: [email protected] Sujing Sun ‡ Affiliations: 1 Wound Healing and Cell Biology Laboratory, Institute of Basic Medicine Science, College of Life Science, Chinese PLA General Hospital, 28 Fuxing Road, Beijing 100853, P.R.China. 2 Key Research Laboratory of Tissue Repair and Regeneration of PLA, and Beijing Key Research Laboratory of Skin Injury, Repair and Regeneration, First Affiliated Hospital to the Chinese PLA General Hospital, 51 Fucheng Road, Beijing 100048, P.R.China E-mail: [email protected] Zhijun Geng Affiliations: Key Research Laboratory of Tissue Repair and Regeneration of PLA, and Beijing Key Research Laboratory of Skin Injury, Repair and Regeneration, First Affiliated Hospital to the Chinese PLA General Hospital, 51 Fucheng Road, Beijing 100048, P.R.China E-mail: [email protected] Wei Sheng Affiliations: Wound Care Center, Institute Of Basic Medicine Science, College Of Life Science, Chinese PLA General Hospital, 28 Fuxing Road, Beijing 100853, P.R.China. E-mail: [email protected] Runkai Chen 1 Environment ACS Paragon Plus

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Affiliations: 1 Tianjin Medical University, No. 22, Qixiangtai Road, Heping District, Tianjin 300070, P.R.China 2 Key Research Laboratory of Tissue Repair and Regeneration of PLA, and Beijing Key Research Laboratory of Skin Injury, Repair and Regeneration, First Affiliated Hospital to the Chinese PLA General Hospital, 51 Fucheng Road, Beijing 100048, P.R.China E-mail: [email protected] Kui Ma Affiliations: Key Research Laboratory of Tissue Repair and Regeneration of PLA, and Beijing Key Research Laboratory of Skin Injury, Repair and Regeneration, First Affiliated Hospital to the Chinese PLA General Hospital, 51 Fucheng Road, Beijing 100048, P.R.China E-mail: [email protected] Xiaoyan Sun* (corresponding author) Affiliations: Wound Healing and Cell Biology Laboratory, Institute of Basic Medicine Science, College of Life Science, Chinese PLA General Hospital, 28 Fuxing Road, Beijing 100853, P.R.China. E-mail: [email protected] Phone：+86-010-66867390 Xiaobing Fu* (corresponding author) Affiliations: 1 Wound Healing and Cell Biology Laboratory, Institute of Basic Medicine Science, College of Life Science, Chinese PLA General Hospital, 28 Fuxing Road, Beijing 100853, P.R.China. 2 Key Research Laboratory of Tissue Repair and Regeneration of PLA, and Beijing Key Research Laboratory of Skin Injury, Repair and Regeneration, First Affiliated Hospital to the Chinese PLA General Hospital, 51 Fucheng Road, Beijing 100048, P.R.China E-mail: [email protected] Phone：+86-010-6686739

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Graphical abstract：：



Abstract Mesenchymal stem cells (MSCs) play an important role in the cutaneous wound healing; however, the functional mechanisms involved in the healing process are poorly understood. A series of studies indicate that keratinocytes which migrate into the wound bed rely on an epithelial-mesenchymal transition (EMT)-like process to initiate re-epithelialization. We therefore examined whether bone marrow-derived MSCs (BMSCs) could affect biological behavior and induce EMT-like characteristics in the human epidermal keratinocytes (HEKs) and in the immortalized human keratinocyte cell line HaCaT cells; and we investigated the signaling pathways of BMSC-mediated phenotypic changes. By assessing the expression of EMT-related markers including E-cadherin, α-SMA and Snail family transcription factors by β2-adrenergic receptor (β2-AR) blockage using ICI-118,551, a β2-AR selective antagonist, or β2-AR small interfering RNA (siRNA), we showed an involvement of β2-AR signaling in the induction of EMT-like alterations in human keratinocytes in vitro. β2-AR signaling also affected collective and individual cell migration in human keratinocyte cell lines, which was attenuated by administration of ICI-118,551. Treating the cells with BMSC-conditioned media (BMSC-CM) not only recapitulated the effect of isoproterenol (ISO) on cell migration, but also induced the expression of β2-AR and a panel of proteins associated with mesenchymal phenotype in HEKs and HaCaT cells. Similarly, blockade of the β2-AR by either ICI-118,551 or β2-AR siRNAs reversed both responses of the epidermal keratinocyte cell lines relative to BMSC-CM exposure. These results were further verified in our vivo findings and indicated that the exogenous application of MSCs promoted cutaneous wound healing and endowed the keratinocytes surrounding the wound area with an increased migratory phenotype through activation of β2-AR signaling. Our findings suggest a biochemical mechanism underlying the function of MSCs in wound re-epithelization, which provide a reliable theoretical basis for the wide application of MSCs in the treatment of chronic wounds. Keywords mesenchymal stem cell, β2-adrenergic receptor, epithelial mesenchymal transition, cutaneous wound healing


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1. Introduction The skin is a powerful barrier to defend our body against a variety of environmental insults and helps to maintain fluid balance within the body. The skin also performs a wide range of functions, including sensation, heat regulation, synthesis, absorption, as well as control of evaporation. Skin diseases, such as deficient wound healing, can lead to loss of function and decreased quality of life, and are a signiﬁcant cause of morbidity. In developed countries, it has been reported that about 1 to 2% of the population will experience a chronic wound during their lifetime1, 2. In the US, an estimated 18% of diabetic patients over the age of 65 suffer from nonhealing foot ulcers3. Worldwide, a lower limb or part of a lower limb is lost every 30 seconds as result of diabetic wound infection. Therefore, chronic wounds represent a major challenge for the clinician and wound care specialist. Understanding and addressing the biological mechanisms of cutaneous wound healing will offer new hope for clinical treatment, resulting in improved patient quality of life and reduced healthcare costs. MSCs are an outstanding tool for cell therapy applications, not only because of their remarkable plasticity, but also due to their ability to home and engraft into damaged tissues4 and low immunogenicity5. With the expansion of MSC research, its potential role in cutaneous wound healing has also been gradually revealed. Several pre-clinical and clinical studies have shown that autologous or allogeneic MSCs are safe and therapeutic in the treatment of chronic wounds6-8, burn injuries9, 10, surgical wounds11, 12, and limb ischemia13. These cells can regulate the function of inflammatory cells, such as macrophages, neutrophils, and T cells, so as to hasten the healing of wounds by triggering an anti-inflammatory response14. Subsequently, MSCs can be directed to differentiate into multiple skin cell lineages including keratinocytes15, adipocytes16, and endothelial cells (ECs)17, and secrete a variety of cytokines to promote wound re-epithelialization and limit excessive scarring14, 18-20. Furthermore, MSCs, in response to the host environment, can be recruited to the site of injury to induce neovascularization21, increase cell migration and proliferation22, and affect the metabolic activity of host cells and tissues23. Moreover, it has been shown that psychological stress hormones are involved in MSC-based wound healing24, with improvement of the re-epithelization process in colonic mucosa25.



The skin is an extremely sensitive neuroendocrine organ. A series of studies have shown that human and rodent skin expressed the genes for corticotrophin-releasing factor (CRF), urocortins, and pro-opiomelanocortin (POMC), with subsequent production of the respective peptides26,

27

. Expression of these elements is organized into functional, cell type-specific

regulatory loops with a structural hierarchy equivalent to that found in the central hypothalamic– pituitary–adrenal axis (HPA axis)

26

. The local cutaneous HPA axis contributes to the central

neuroendocrine response, which regulates skin homeostasis and interferes with the wound healing process28,

29

. The regulation of the neuroendocrine system in skin physiology is

intriguing30. For example, some studies reported that β1- and β2- adrenoceptor blockade delayed cutaneous wound healing in rats31, while others reported that β2-AR antagonists increased keratinocyte migration by preventing the binding of endogenously or systemically synthesized epinephrine32. Given its pleiotropic effects on the structure and cellular components of the skin, it makes sense that regulation of the skin neuroendocrine network and its mediated signaling pathways provides a potential target for accelerating wound healing and skin tissue regeneration. In this study, we hypothesized that MSCs accelerated wound healing and keratinocyte migration by interfering with β2-AR signaling. To examine our hypothesis, we first detected the expression of EMT-associated proteins in HEK and HaCaT epidermal keratinocyte cell lines at both mRNA and protein levels after isoproterenol stimulus with either ICI-118,551 or β2-AR siRNA treatment. Our results showed an involvement of β2-AR signaling in the induction of EMT-like alterations in human keratinocytes in vitro. β2-AR signaling also affected the healing of scratch wounds in confluent keratinocyte cultures, and resulted in an increase in the individual cell motility as measured by the in vitro single cell migration assay. Given that MSCs enhanced the cutaneous wound healing via paracrine interactions with the cell components in wound area33, 34, we next investigated the effect of BMSC-CM on the biological changes of human keratinocytes, and surprisingly found that BMSC-CM administration recapitulated the effect of isoproterenol on cell migration and induced the expression of β2-AR and a panel of proteins associated with the mesenchymal phenotype in both HEKs and HaCaT cells. These results were further validated in our vivo findings that the application of exogenous MSCs led to an acceleration of wound closure and re-epithelization by promoting EMT-like phenotypic changes in wounded tissues following activation of β2-AR signaling.


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2. Experimental Section 2.1. Cell culture and treatment The human epidermal keratinocytes (HEKs) and HaCaT cells were purchased from the Thermo Fisher Scientific, Inc. and China Infrastructure of Cell Line Resources, respectively. The cells were incubated in basic EpiLife medium (Invitrogen) supplemented with 0.06 mM Ca2+ and 1% EpiLife deﬁned growth supplement (Invitrogen). Human BMSCs (Cyagen Biosciences) were cultured in low-glucose DMEM containing 10% fetal bovine serum (FBS; Invitrogen) and 1% penicillin/streptomycin (Sigma-Aldrich) in a humidified chamber with 5% CO2. For treatment with β-adrenergic receptor (β-AR) agonists, HEKs and HaCaT cells were starved overnight, then treated with 5µM isoproterenol (Sigma) for indicated time points. For β2-AR antagonist treatment, the cells were first treated with 10µM ICI-118,551 (Sigma), a selective β2-AR antagonist, for 1hr before isoproterenol stimulation. For the preparation of BMSC-CM, confluent BMSC were washed with phosphate-buffered saline (PBS), transferred to basic EpiLife medium (Invitrogen) and incubated for 48 hr. The culture supernatants were collected and then concentrated by ultrafiltration with a 3kDa molecular weight cut-off concentrating columns (Millipore) according to the manufacturer’s protocol. For PKA antagonist treatment, HEKs and HaCaT cells were first treated with 10µM H89 (Abcam), a selective PKA antagonist, for 1hr before BMSC-CM stimulation. The effects of differential dosage of ICI-118,551 on β2-AR activity in epidermal keratinocytes were demonstrated in supplementary Fig. 1A and B, and the characterization and relative cell viability of BMSCs were shown in supplementary Fig. 1C and D. 2.2. Constructs and cell transfection Pre-designed siRNAs directed to β2-ARs (si-β2-AR) and non-targeting control siRNAs were purchased from GenePharma, which were used at a concentration of 50 nM and contained the following sequences (5′ to 3′): si-β2-AR1-sense: CCACGACGUCACGCAGCAATT, si-β2-AR1antisense:

UUGCUGCGUGACGUCGUGGTT;

si-β2-AR2-sense:

CCUAGCGAUAACAUUGAUUTT, si-β2-AR2-antisense: AAUCAAUGUUAUCGCUAGGTT; si-β2-AR3-sense:

GCUCCAGAAGAUUGACAAATT,

UUUGUCAAUCUUCUGGAGCTT;

negative


si-β2-AR3-antisense: control

siRNA-sense:


UUCUCCGAACGUGUCACGUTT,

negative

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control

siRNA-antisense:

ACGUGACACGUUCGGAGAATT. The HEKs and HaCaT cells were plated in basic EpiLife medium (Invitrogen) in 6-well plates. When the cells reached a confluence of 60-70%, transfection was carried out by using INTERFERin® in vitro siRNA/miRNA Transfection Reagent (Polyplus Transfection) according to the manufacturer's protocol. All transfections were carried out in triplicate and repeated at least three times. 2.3. Quantitative real-time PCR analysis Total cellular RNA was isolated with RNAgents Total RNA isolation system (Promega) with DNase I (Invitrogen) treatment. cDNA was synthesized using PrimeScript RT reagent Kit (TaKaRa). Real-time PCR was performed using the SYBR ○ R Green I on GoTaq ○ R qPCR Detection System (Promega) according to the manufacturer’s recommendations. The results were analyzed using the comparative threshold cycle method with GAPDH as an internal control. The primer sequences used for gene amplification are listed in Table 1. 2.4. Western blot analysis Total protein was isolated from the cells using a RIPA buffer (Sigma). The freshly dissected skin tissue was snap frozen in liquid nitrogen, then homogenized using electric grinder in the RIPA. Whole cell lysates were prepared, separated by SDS-PAGE, and transferred to PVDF membranes. After blocking with 5% BSA, blots were probed with the following primary antibodies overnight at 4 ℃: anti-β2-AR rabbit polyclonal antibody (1:1000, Abcam), E-cadherin (4A2) mouse mAb (1:1000, CST), anti-alpha smooth muscle actin mouse monoclonal antibody (1:1000, Abcam), rabbit polyclonal to SNAIL+SLUG antibody (1:1000, Abcam), Slug (C19G7) Rabbit mAb (1:1000, CST), phospho-CREB (Ser133) (87G3) rabbit mAb (1:1000, CST), mouse monoclonal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:1000, Abcam). The membranes were washed with TBST and then incubated with horseradish peroxidaseconjugated secondary antibodies (1:1000, CST) for 2hr at room temperature. The immunoreactive bands were detected by enhanced chemiluminescence (ECL) kit (Solarbio) and imaged using the ImageQuant LAS 4000 system (GE Healthcare Bio-Sciences). 2.5. Scratch wound assay


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HEKs and HaCaT cells were grown to confluence in 6-well plates. After starvation overnight, the cells were treated with 10 µg/ml Mitomycin C (Millipore) for 1 hr to inhibit proliferation and wounded with a pipette tip. Then, the wounded monolayers were washed three times and treated with 0 or 5 µM isoproterenol (Sigma) for an additional 6hr. For β2-AR blockage, wounded monolayers were preincubated with 10 µM ICI-118,551 (Sigma) for 1hr before exposure to isoproterenol stimulation. For BMSC-induced cell migration evaluation, the starved cells were switched to BMSC-CM for indicated time points with or without ICI-118,551 (Sigma). Scratch-wound closure was recorded over 24 hr using time-lapse phase-contrast microscopy at a frame rate of one picture every 60 min with a 10× objective. The relative cells migration to the cell-free area was measured by using ImageJ 1.42 software. 2.6. Single cell migration assay Starved HEKs and HaCaT cells were seeded at a density of 1×104 cells/well/1ml medium in 24-well plates. After initial attachment onto well surfaces, the cells were incubated with either basic EpiLife medium (Invitrogen) alone or EpiLife containing 5µM isoproterenol (Sigma). For β2-AR blockage, the cells were preincubated with 10µM ICI-118,551 (Sigma) for 1hr before exposure to isoproterenol stimulation. For BMSC-induced cell migration evaluation, the starved cells were switched to BMSC-CM with or without ICI-118,551 (Sigma). The plates were loaded into the live cell chamber of the Operetta High Content Imaging System (Perkin Elmer), which was pre-programmed to retain the chamber at optimal growth conditions (37 °C; 5% CO2). Operetta software was used for image acquisition. Individual cell migration was monitored over a 24-hr period using time-lapse phase-contrast microscopy on a multi-field acquisition at a frame rate of one picture every 10 min with a 10× objective. The resulting time-lapse image sequences were batch analyzed using the Harmony High Content Imaging and Analysis Software (Perkin Elmer). 2.7. Cell viability assay HEKs and HaCaT cells (1 × 103/well) were plated in 96-well plates in quadruplicate respectively, and incubated at 37°C in 5% CO2 humidified atmosphere. After a 24hr starvation, the growth medium of the cells was replaced with the BMSC-CM for indicated time points. The relative cell viability was then detected using the MTT (Sigma) assay following the



manufacturer's recommendations, and the absorbance was measured at 490 nm using a BIORAD iMark microplate reader. The cells not treated with BMSC-CM were used as controls. 2.8. Enzyme-Linked Immunosorbent Assay (ELISA) The filtered BMSC-conditioned medium was used to determine the catecholamine levels by a Human Catecholamine ELISA Kit (AMEKO) according to the manufacturer’s instructions. Basic EpiLife medium without BMSCs was used as a control. 2.9. In vivo wound healing model 8-weeks old male C57BL/6J mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were chosen for the experiment. For analysis of the epithelialization process, the excisional wound splinting model was generated to prevent wound contraction as described previously35, In brief, after hair removal from the dorsal surface and anesthesia, 6-mm full thickness excisional skin wounds were created on the dorsal skin of the mice. Then, the mice were randomly assigned (n=5

for each group) to receive one of the following treatment options: human BMSCs or vehicle (saline solution). Human BMSCs (2×106 cells in 200 µl of vehicle) were injected once every 3 days with 160 µl for subcutaneous injection around the wound at four sites and 40 µl for topical application on the wound bed. Similarly, 200 µl saline solution was intradermally injected in vehicle around the wound and wound bed. A donut-shaped silicone splint was positioned so that the wound was centered within the splint. An immediate-bonding tissue adhesive (3M) was used to fix the splint to the skin, followed by interrupted sutures to stabilize its position, and Tegaderm (3M) was placed over the wounds. The animals were housed individually. We tested the adhesive on the skin in mice prior to this experiment and did not observe any skin irritation or allergic reaction.

Meanwhile, for quantitative evaluation of the expression levels of EMT-associated markers during skin wound healing, one 10-mm circular diameter full-thickness wound was placed on the dorsal shaved skin of the mice as previously described36. The animals were randomly divided into two groups (n=15 for each group) which received either human BMSCs or vehicle by both subcutaneous injection around the wound and topical application to the wound bed as mentioned above. Normal murine skin tissues from an adjacent area were used as blank controls. All animal

procedures were performed in accordance with institutional guidelines. 2.10. Wound Closure Analysis


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The wound areas were analyzed in a timely manner on days 0, 2, 4, 6, 8 and 10. The percentage of wound closure was calculated using the formula [(area of original wound - area of actual wound)/(area of original wound)] x 100. Mice were sacrificed at indicated days, when skin biopsies from each group were harvested for further analysis. 2.11. Immunofluorescence staining Mouse dorsal skin samples were fixed in 10% formaldehyde for 24-48hr followed by paraffin embedding and microtome sectioning (4-µm thickness). Slides were then stained with hematoxylin and eosin (H&E). To perform immunofluorescence staining, frozen sections of the mouse skin biopsies were prepared. The sections were fixed in 4% paraformaldehyde for 30 min, and then permeabilized in 0.25% Triton X-100 (Sigma-Aldrich) in PBS for 15 min. After blocking with 5% normal goat serum, the sections were stained with anti-β2-AR rabbit polyclonal antibodies (1:200, Abcam), anti-E-cadherin rabbit monoclonal antibody (1:500, Abcam), anti-alpha smooth muscle actin mouse monoclonal antibody (1:200, Abcam), antiCytokeratin 14 rabbit monoclonal antibody (1:1000, Abcam) overnight at 4 °C. The appropriate Alexa-Fluor-conjugated secondary antibodies (1:200, Abcam) were used, and DNA was counterstained using DAPI (Vector Laboratories). Fluorescence images were collected with a laser scanning confocal microscope (SP8, Leica). This experiment was repeated in duplicate. 2.12. Statistical analysis All values are presented as the means ± standard deviation (S.D.). The data conform to the normal distribution and the variance is homogeneous. Statistical analysis was performed using SPSS 20.0 software. Comparisons between two groups were analyzed by Dunnett's T test, and comparisons between more than two groups were analyzed by One-way ANOVA. Probability (P) values

Bone marrow-derived mesenchymal stem cells promoted cutaneous

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