Loss of Atg7 in Endothelial Cells Enhanced Cutaneous Wound Healing in a Mouse Model

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Available online at www.sciencedirect.com

ScienceDirect journal homepage: www.JournalofSurgicalResearch.com

Loss of Atg7 in Endothelial Cells Enhanced Cutaneous Wound Healing in a Mouse Model Ke-Cheng Li, MD, PhD,a,1 Chun-Hui Wang, MD,b,1 Jing-Jiang Zou, MD,a Chen Qu, MD, PhD,c Xing-Li Wang, MD, PhD,e Xing-Song Tian, MD,f Hong-Wei Liu, MD, PhD,a,* and Taixing Cui, MD, PhDd,** a

Department of Plastic Surgery,The First Affiliated Hospital of Jinan University,Guangzhou, Key Laboratory for Regenerative Medicine, Ministry of Education,Institute for New Technologies Plastic Surgery of Jinan University, Guangzhou, Guangdong, China b Surgical Intensive Care Unit, Shandong Provincial Hospital affiliated to Shandong University, Jinan, Shandong, China c Ultrasound Diagnosis and Treatment Department, Shandong Provincial Hospital affiliated to Shandong University, Jinan, Shandong Province, China d Department of Cell Biology and Anatomy, University of South Carolina School of Medicine, Columbia, South Carolina e Shandong University Qilu Hospital Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital of Shandong University, Jinan, Shandong, China f Department of Breast and Thyroid Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong, China

article info

abstract

Article history:

Background: Emerging evidence has linked autophagy to skin wound healing; however, the

Received 25 January 2019

underlying cellular and molecular mechanisms remain poorly understood. The present

Received in revised form

study was designed to determine the role of autophagy in endothelial cell (EC)-mediated

19 November 2019

skin wound healing in mice.

Accepted 3 December 2019

Methods: Autophagy-related gene (Atg7) in mouse ECs was inactivated by the Cre-loxP

Available online xxx

system under the control of an EC-specific VE-Cadherin (Cdh5) promoter (Atg7EC
/
mice). Full-thickness skin wounds were created on the dorsum of wild-type (WT), Cdh5-

Keywords:

Creþ, floxed Atg7 (Atg7F/F), and Atg7EC
/
mice. Autophagic activity was determined by

Autophagy

autophagic flux assay in the primary culture of ECs isolated from these mice. The wound

Wound healing

re-epithelialization and angiogenesis was examined by histological analyses. The angio-

Endothelial cell

genic activity of ECs was evaluated by tube formation assay in vitro. EC proliferation was

Paracrine

examined by a cell count CCK-8 kit. EC-originated intercellular communication with

Angiogenesis

dermal fibroblasts and keratinocytes was assessed by measuring the effect of EC conditional medium on the growth of keratinocytes and fibroblasts. The levels of VEGF, EGF, bFGF in EC conditional medium were measured by ELISA.

* Corresponding author. Department of Plastic Surgery, The First Affiliated Hospital of Jinan University, Guangzhou, Key Laboratory for Regenerative Medicine, Ministry of Education, Institute for New Technologies Plastic Surgery of Jinan University, Guangzhou, Guangdong, 510632, China. Tel.: þ86 20-38688163; fax: þ86-20-3868-8446. ** Corresponding author. Department of Cell Biology and Anatomy, University of South Carolina School of Medicine, Columbia, SC 29209. Tel.: þ1(803)-216-3804; fax: þ1-803-216-3846. E-mail addresses: [email protected] (H.-W. Liu), [email protected] (T. Cui). 1 These authors contribute equally to this work. 0022-4804/$ e see front matter ª 2019 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jss.2019.12.004

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Results: Autophagy deficiency in ECs markedly enhanced the re-epithelialization and the wound closure during skin wound healing. However, it has minimal impact on angiogenesis in the wounded skin. Notably, autophagy deficiency in ECs did not affect their proliferation and migration or angiogenic activity per se but enhanced the EC conditional medium-induced proliferation and migration of keratinocytes and fibroblasts. Conclusions: These results demonstrate for the first time an inhibitory role of autophagy in the EC-originated paracrine regulation of skin wound healing. ª 2019 Elsevier Inc. All rights reserved.

Introduction Autophagy (Greek for “self-eating”) is an evolutionarily conserved pathway that targets cytoplasmic components to the lysosome for degradation. Autophagy has been classified into three different types depending on the means by which the target is delivered into the lysosome: (1) macroautophagy that is mediated by formation of double-membrane-bound vesicles termed the autophagosome which engulfs a portion of cytoplasm or cytoplasmic organelles and delivers its content to lysosomes by fusion with the lysosome; (2) microautophagy by which the cytoplasmic substances are directly engulfed and internalized by lysosomes; (3) chaperonemediated autophagy that involves the direct translocation of cytosolic proteins across the lysosomal membrane by chaperon proteins.1-3 Macroautophagy (hereafter referred as autophagy) is thought to be the major type of autophagy, and it is critical for the clearance of dysfunctional proteins and damaged organelles in the cell thus maintaining physiological homeostasis. In addition, autophagy generally acts as an important adaptive mechanism for cell survival. Moreover, deregulated autophagy is detrimental to the cell, particularly in some pathophysiological settings, thereby contributing to the pathogenesis of numerous diseases.4 However, the precise pathophysiological relevance of autophagy remains poorly understood. Skin wound healing is a complex series of reactions and interaction among dermal cells and humoral mediators which are characterized by several overlapping phases including immediate humoral factor release, inflammatory responses, angiogenesis, proliferation, re-epithelialization, and remodeling.5,6 The recruitment of inflammatory cells at the early phase is important for the transition into the later phase, while angiogenesis, a process of new blood vessel formation characterized by the sprouting of preexisting blood vessels, is believed to be one of the major biological responses supporting cutaneous wound healing.6,7 To date, the cellular and molecular mechanisms of skin wound healing are still far from a comprehensive understanding. Given the wellestablished role of autophagy in regulating inflammation, angiogenesis, and cellular growth,8-10 it is conceivable that autophagy may play a critical role in skin wound healing. However, the paucity of studies regarding the autophagymediated wound healing is controversial. A previous report has suggested that autophagy may suppress resident T cell function thereby delaying the wound repair of sterile fullthickness skin wounds.11 By contrast, the other studies have proposed that autophagy may suppress inflammation thus acting as an adaptive response for burn skin wound

healing.12,13 Because these studies lack the genetic targeting approach, the precise role of autophagy in skin wound healing remains unclear. To date, the direct evidence for a mediator role of autophagy in skin wound healing is missing. Therefore, in the present study, we used the mice with endothelial cellespecific knockout of Atg7, an E-1 like ubiquitin-activating enzyme which activates autophagy by inducting autophagosome formation,14 and determined the impact of autophagy deficiency in endothelial cells on skin wound healing. We found that autophagy in endothelial cells is dispensable for angiogenesis but most likely required for the control of endothelial celleoriginated intercellular communications in the skin, thereby influencing skin wound healing.

Materials and methods Animals Breeding pairs of floxed Atg7 (Atg7F/F) mice in C57BL/6J background (Stock#: RBRC 02231) and VE-Cadherin (Cdh5)-Cre mice in C57BL/6J background (Stock#: 006137) were purchased from Riken BioResource Center, Japan and JAX, respectively. Animals were housed in the institute’s animal facility under standard conditions (a 12-h light/dark cycle and an average temperature of 26 C) and given to standard laboratory food and water ad lib. Atg7F/F mice were crossed with Cdh5-Creþ/þ mice to generate homozygous Atg7 endothelial-specific deletion (Atg7F/F:Cdh5-Creþ, i.e., Atg7EC
/
) mice (Fig. 1A). Genotypes of the animals were determined by PCR amplification of genomic DNA obtained from the tail. The PCR products were resolved on 1% agarose gel. The genotypes of mice were verified by examining the size of the PCR products: wild-type (WT) (1500 bp), Atg7F/F (550 bp), EC Atg7
/
(550 bp and 2100 bp), EC Atg7þ/
(550 bp, 1500 bp, and 2100 bp), and Cdh5Creþ (324 bp for internal control gene of interleukin-2 precursor, and 100 bp for Cre). Primers for genotyping are: 50 -TGG TGC TAC TTC TGC AAT GAT GT-30 (sense for Atg7F/F), 50 -CAG GAC AGA GAC CAT CAG CTC CAC-30 (antisense for Atg7F/F), 50 TTA GCA CAG GGA ACA GCG CTC ATG G-30 (antisense for Atg7F/F); 50 -GAA CCT GAT GGA CAT GTT CAG GGA-30 (sense for Cdh5-Creþ), 50 -CAG AGT CAT CCT TAG CGC CGT AAA-30 (antisense for Cdh5-Creþ), 50 -CTA GGC CAC AGA ATT GAA AGA TCT-30 (sense for the internal control), and 50 -GTA GGT GGA AAT TCT AGC ATC ATC C-30 (antisense for the internal control) (Fig. 1B). All animal experiments were conducted in accordance with the National Institutes of Health (NIH) Guidelines for Care and Use of Laboratory Animals and

li et al  role of atg7 in wound healing

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B

C

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Fig. 1 e Generation of Atg7 endothelial cellespecific knockout (Atg7ECL/L) mice. (A) A scheme of breeding approach for the generation of Atg7ECL/L mice. (B) Genotypes of the animals were determined by PCR amplification of genomic DNA obtained from the tail. The genotypes of mice were verified by examining the size of the PCR products: wild-type (WT) (1500 bp), Atg7F/F (550 bp), EC Atg7L/L (550 bp and 2100 bp), EC Atg7D/L (550 bp, 1500 bp, and 2100 bp), and Cdh5-CreD (324 bp for internal control gene of interleukin-2 precursor, and 100 bp for Cre). (C) Western blot analysis of Atg7 expression in the primary culture of myocardial endothelial cells isolated from WT, Atg7F/F, Cdh5-CreD (CreD), and Atg7ECL/L mice (n [ 2). (D) Autophagic flux assay. Cultured myocardial endothelial cells isolated from WT, Atg7F/F, CreD, Atg7ECD/L, and Atg7ECL/L mice were treated with or without BafA1 (10 nM) for 4 h and then subject to Western blot analysis of LC3. Left top panel: representative immunoblots of LC3-I & II. Lower and right panels: densitometric analysis of LC3-I & II expression. n [ 4. *P < 0.05 versus BafA1 (-) in the same group. #P < 0.05 versus other groups. (Color version of figure is available online.)

approved by the Institute Animal Care and Use Committee (IACUC) at Shandong University, China, and the University of South Carolina.

Wound protocol and analysis of wound area Male mice at ages of 8-9 wk were anesthetized by intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/ kg). Hair on the back was shaved and swabbed with povidoneiodine and 75% ethanol. A full-thickness excisional circular wound (6 mm in diameter) was made using a biopsy punch on the dorsum of each mouse, left unsutured without dressing. The wound on the dorsum of each mouse was photographed every day and analyzed by ImageJ software (NIH, Maryland). Wounded skin, together with a margin of healthy skin, was

excised by an 8-mm diameter biopsy punch at days 3 and 10 after wounding and then subjected to the preparation of 5mm-thick paraffin tissue sections. Unhealed wound area was calculated as a percentage of the original wound size by using the wound images. The formula for the calculation is as follows: wound area (%) ¼ (wound size/original wound size)  100%. Five to seven mice for each group were analyzed at each time point.

Isolation and culture of mouse myocardial endothelial cells as well as dermal fibroblasts and keratinocytes Mouse myocardial endothelial cells were isolated from the heart of WT, Cdh5-Creþ, Atg7F/F, Atg7þ/
, Atg7
/
mice at ages of 6-9 wk as previously described with a few modulations.15

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Briefly, the mouse heart was minced into 3  3 mm pieces and digested in collagenase I (0.1%) for 30 min at 37 C on a rotator. The digestion was neutralized by same amount of DMEM supplemented with 10% FBS. The digested solution was filtered through first 200-mesh (74 mm) and then 400-mesh (37 mm) to discard large fragments of connective tissues. The filtrate was centrifuged at 250  g for 10 min. The pellet was resuspended with 1.5 mL 40% Percoll (Sigma-Aldrich) in PBS, overlaid consecutively by 1.5 mL 20% Percoll and 1.5 mL PBS, and centrifuged at 400  g for 5 min. The generated turbid middle layer was collected and diluted with plain DMEM. The cell suspension was centrifuged at 200  g for 5 min, and the cell pellet was collected and washed once in DMEM by centrifugation at 200  g for 5 min. Finally, the cell pellet was resuspended in Endothelial Cell Medium (ECM; Cat#: 1001, ScienCell Research Laboratories) supplemented with 1% Endothelial Cell Growth Supplement (ECGS; Cat#: 1052, ScienCell Research Laboratories) for culture in a CO2 incubator at 37 C. The purity of cultured mouse myocardial endothelial cells was up to 80%, which were determined by CD31 and von Willebrand factor staining (data not shown). Cultured cells at passage 5-8 were used for experiments. Mouse dermal fibroblasts and keratinocytes were isolated and cultured as previously described.16,17 Briefly, mouse dermal fibroblasts were isolated from neonatal WT mice and cultured in DMEM supplemented with 10% FBS. Fourth passage cells were used. Keratinocytes were isolated from these neonatal mice and cultured in defined keratinocyte serum-free medium (dKSFM) (Invitrogen, Carlsbad, CA) supplemented with 10 ng/mL epidermal growth factor and 10
10 M cholera toxin and penicillin/streptomycin (Sigma, Munich, Germany). Keratinocytes in their fourth passage were used in this study. All migration experiments were performed under the same experimental conditions.

Proliferation, wound healing, and tube formation assay in vitro Cells (2000 per well) were seeded into 96-well culture plates. The cell number was measured by a CCK-8 kit (Dojindo Molecular Technologies, Inc) at 1 d, 3 d, and 7 d after seeding. The experiment (n ¼ 6) was repeated at least three times. Cells were seeded into 6-well plates. A wound was generated by scratching endothelial cells at a confluent state. The wound area was tracked down at 0, 12, and 24 h after the scratch. Wound areas were measured using Image-Pro Pus 6.0. The decrease in wound areas reflects the migration of the cells. The experiment (n ¼ 3) was repeated at least three times. Cells were seeded at the density of 1.5  105 cells/well into 48-well plates precoated with 100 mL/well Matrigel (BD, Biosciences). Tube formation was observed using an inverted microscope and quantified using ImageJ software (NIH, Maryland) after 12 h. The experiment (n ¼ 3) was repeated at least three times.

Western blot Western blot was performed as previously described.18 In brief, equal amounts of cell lysates were separated by SDSPAGE (12%) and transferred onto polyvinylidene difluoride

membranes. Membranes were blocked with 5% (w/v) nonfat milk dissolved in TBST for 1 h at room temperature and then incubated with the blocking solution containing primary antibody (anti-Atg7, anti-LC3A/B, and anti-beta-actin) overnight at 4 C. After washing three times with TBST, the blot was incubated with a second antibody. Bands of identity were visualized with an enhanced ECL reagent. Quantification of the luminosity of each identified protein band was performed using a densitometric scanner (AlphaView, Cell Biosciences).

Statistical analysis Values are expressed as mean  SEM in the text and figures. The data were analyzed by one-way ANOVA. Differences between two groups were evaluated for statistical significance using Student’s t-test. A value of P < 0.05 was considered to be statistically significant.

Results Generation of Atg7 endothelial cellespecific knockout (Atg7EC
/
) mice Atg7 endothelial cellespecific knockout (Atg7EC
/
) mice were generated by crossing floxed Atg7 (Atg7F/F) mice with Cdh5Cre mice as depicted in Figure 1A. To verify the efficiency of Atg7 knockout in the endothelial cells of Atg7EC
/
mice, we determined the Atg7 protein expression in the cardiac endothelial cells isolated from mice with different genotypes. As shown in Figure 1C, there was an approximately 50% decrease in the protein expression of Atg7 in the endothelial cells of heterozygote Atg7ECþ/
mice, and an about 90% decrease of Atg7 protein expression in the endothelial cells of homozygote Atg7EC
/
mice, compared to WT mice. The Atg7 protein expression levels are similar in the endothelial cells of WT, floxed Atg7 (Atg7F/F), and Cdh5-Creþ mice (Fig. 1C). During the autophagic response, LC3-I, a 16-kDa homologue of Atg8 in yeast, is conjugated with phosphatidylethanolamine (PE) to become LC3-II (LC3-PE), an active isoform that migrates from the cytoplasm to the elongating autophagosome membrane and remains on completed autophagosomes until fusion with the lysosomes.2,3,19 The ratio of LC3-II to LC3-I may be used as a parameter to monitor autophagosome formation. The protein abundance of LC3-II usually reflects the steady level of autophagosomes, which is dependent on a balance between autophagosome synthesis and autophagosome clearance via lysosomes.2,3,19 Although the actual molecular weight (MW) of LC3-II is larger than that of LC3-I, LC3-II (apparent MW is 14-kDa) migrates faster than LC3-I in SDS-PAGE because of extreme hydrophobicity of LC3-II.2,3,19 Because Atg7 is critical for LC3-I conjugation and autophagosome formation, loss of Atg7 should lead to downregulation of a steady level of LC3-II and upregulation of LC3-I in endothelial cells as previously reported.14 Indeed, we observed that the expression of LC3-I and LC3-II proteins was similar in the isolated cells from WT, Cdh5-Creþ, and Atg7F/F mice and the relatively higher level of LC3-II indicates steady activation of autophagy in these cells (Fig. 1D). However, the protein expression of LC3-I was markedly increased while the LC3-II

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wound healing.14 Because the steady levels of LC3-I and LC3-II as well as autophagic flux in the endothelial cells isolated from WT and Atg7F/F mice are similar (Fig. 1D), the flox insertion is not likely to affect autophagy in endothelial cells. These results suggest that the delayed skin wound healing in Atg7F/F mice may be caused by a yet unknown mechanism independent of endothelial autophagy. Although the underlying mechanism remains to be determined, these results revealed that Atg7F/F mice have to be used as the control for the experiments using Atg7EC
/
mice.

expression was significantly reduced in the isolated cells from Atg7EC
/
mice, compared with that from WT, cdh5-Creþ, and Atg7F/F mice (Fig. 1D). These results indicate an impaired autophagy in the endothelial cells isolated from Atg7EC
/
mice. To further confirm this notion, we measured autophagic flux, a more accurate assessment of autophagy that indicates the capacity of autophagic clearance in the cell,2,3,19 in the endothelial cells isolated from WT, Cdh5-Creþ, Atg7F/F, and Atg7EC
/
mice. Autophagic flux was assessed by measuring the accumulation of LC3-II induced by bafilomycin A1 (BafA1), an inhibitor of autophagosome-lysosome infusion as described elsewhere.20 BafA1-induced accumulation of LC3-II was observed in the endothelial cells of WT, Cdh5-Creþ, and Atg7F/F mice but not in the cells of Atg7EC
/
mice (Fig. 1D), demonstrating an impaired autophagy in the endothelial cells of Atg7EC
/
mice. Thus, the conditional Atg7EC
/
mice were successfully generated. The Atg7 endothelial cellespecific knockout did not induce apparent changes in histological morphologies of endothelial celleenriched organs including thoracic aorta, carotid artery, lung, and kidney (Fig. S1), suggesting that loss of Atg7 in endothelial cells has a minimal impact on the development of major organ systems.

The effect of Atg7 endothelial cellespecific knockout on skin wound healing To investigate the effect of Atg7 endothelial cellespecific knockout on skin wound healing, we examined the healing processes of full-thickness skin excisional wounds in Atg7EC
/
mice using Atg7F/F mice as the control. Macroscopic analyses of time-matched Atg7EC
/
versus Atg7F/F control wounds showed that wound closure is markedly accelerated at the indicated time points during the wound repair in Atg7EC
/
mice, with a mean of 85% wound closure achieved by day 10 in Atg7EC
/
mice and a mean of 70% wound closure in Atg7F/F mice (Fig. 3A and B). Wound re-epithelialization as measured by length of epithelial tongue and epithelial gap at day 3 after wounding was significantly faster in Atg7
/
mice compared with Atg7F/F mice (Fig. 3C).

The impact of flox and Cre gene inserts on skin wound healing at the basal level Because any genetic manipulation potentially affects some pathophysiological processes, we determined whether the flox and Cre insertions per se have any impact on skin wound healing in mice. Compared with the WT control, the healing of full-thickness skin excisional wounds was delayed in Atg7F/F mice at day 8 and day 10 after skin injury, but not in Cdh5Creþ/þ mice (Fig. 2). These results indicate that the flox inserts flanked the exon 14 of Atg7 gene slightly suppress skin

The effect of Atg7 endothelial cellespecific knockout on angiogenesis in skin wound healing in vivo and endothelial cell proliferation and migration in vitro To determine whether Atg7EC
/
impairs angiogenesis in skin wound healing, microscopic analyses of CD31 (an established

WT

Atg7F/F

Cre+

6 mm

WT Atg7F/F Cre+

120 80 *

40 0

1

2

3

**

8

Days after wound injury

Days after wound injury

Wound Area (%)

0

1

3

8

10 10

Fig. 2 e The effects of flox and Cre gene insertions on skin wound healing. Left top panel: a schematically depicted excisional skin wound injury in mice. Left lower panel: quantified unhealed skin wound areas in WT, Atg7F/F, and Cdh5CreD/D (CreD/D) mice at indicated times after injury. n [ 5-8 for each group. *P < 0.05; **P < 0.01. Right panel: macroscopic observation of excisional wounds in WT, Atg7F/F, and CreD/D mice at various time points after woundingdrepresentative images. Scale bar are 5 mm. (Color version of figure is available online.)

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A

Atg7F/F

B

Atg7EC-/-

0

**

Wound Area (%)

Days after injury

120 1

3

Atg7F/F Atg7EC-/-

* 80

*

**

40 0 1

8

3

8

10

Days after wound injury

10

c

Length epithelial tongue

Length epithelial tongue

endothelial cells isolated from Atg7F/F and Atg7
/
mice are similar, indicating that autophagy impairment due to the loss of Atg7 has minor impact on endothelial cell proliferation and migration. In fact, it has been documented that suppression of autophagy by disruption of Beclin 1 has no effects on endothelial cell growth and migration under a normal culture condition.21 To further investigate the effects of autophagy impairment on endothelial cell tube formation, the total tube length of the tube-like structure was measured. There was no significant difference of total tube length between Atg7F/F and Atg7
/
mice (Fig. 5A). These results suggest that autophagy impairment in endothelial cells may not affect endothelial cell growth and migration thus having minimal impact on angiogenesis in vivo.

The effect of Atg7 endothelial cellespecific knockout on endothelial cell paracrine regulation

Epithelial gap

Atg7F/F Length epithelial tongue

Length epithelial tongue

Atg7EC-/-

1.4 1.0 0.6 0.2 0.0

**

Epithelial gap (mm)

Length epithelial tongue (mm)

Epithelial gap

2.5 2.0 1.5 1.0 0.5 0.0

p=0.07

Fig. 3 e Morphological analysis of wounded skin in Atg7F/F and Atg7ECL/L mice. (A) Macroscopic observation of excisional wounds in Atg7F/F and Atg7ECL/L mice at various time points after woundingdrepresentative images. Scale bar are 5 mm. (B) Quantified unhealed skin wound areas in Atg7F/F and Atg7ECL/L mice at indicated times after injury. n [ 5-7 for each group. *P < 0.05, **P < 0.01 versus Agt7F/F control in the same group. (C) Upper panel: representative H&E staining of wounded skin tissue sections of Atg7F/F and Atg7ECL/L mice at day 3 after injury. Lower panel: quantified length of epithelial tongue and epithelial gap of wounded skin tissues in Atg7F/F and Atg7ECL/L mice at day 3 after injury. n [ 3. **P < 0.01 versus Agt7F/F control. (Color version of figure is available online.)

endothelial biomarker) staining in wounded skin were performed. There was no significant difference of CD31-positive cells between Atg7F/F and Atg7
/
mice (Fig. 4A), suggesting that loss of Atg7 in endothelial cells hardly affects angiogenesis in skin wound healing. To further confirm the observation, we examined the effect of Atg7 knockout on endothelial cell proliferation and migration using the primary culture of endothelial cells isolated from Atg7F/F and Atg7
/
mice. As shown in Figure 4B and C, the growth rate and migration of

Endothelium has been considered as an “endocrine organ” due to the endothelial cellemediated autocrine and paracrine regulation of intercellular communications in the vasculature.22,23 Although endothelial cells express chemokines which may be responsible for the recruitment of inflammatory cells to the wounded skin,24 there are few studies regarding the intercellular communication between endothelial cells and epidermal fibroblasts in the skin.25,26 Nevertheless, the nature and pathophysiological significance of endothelial celleoriginated communication in skin wound healing remain largely unknown. Intriguingly, we found that the recruitments of macrophages and lymphocytes at 3 d after skin injury were increased in Atg7EC
/
mice compared to Atg7F/F mice (Fig. 6). These results indicate that autophagy deficiency in endothelial cells enhances inflammatory responses at the early phase of skin wound healing, presumably through the regulation of endothelial cell paracrine. Indeed, the conditional medium of cultured Atg7EC
/
endothelial cells significantly enhanced the proliferation and migration of dermal fibroblasts and keratinocytes (Fig. 7 and 8). And the secretion of bFGF, EGF was increased in Atg7EC
/
ECs compared to Atg7F/F ECs, meanwhile the VEGF was not detected (Fig. 5B); these findings reveal that autophagy deficiency in endothelial cells facilitates their paracrine regulation of the acute inflammatory responses as well as the growth of dermal fibroblasts and keratinocytes thereby enhancing skin wound healing.

Discussion Impaired or aberrant skin wound healing eventually lead to major clinical problems. Understanding the precise cellular and molecular mechanisms of skin wound repair in vivo is therefore of utmost importance. In the present study, we have demonstrated that autophagy deficiency in endothelial cells facilitates skin wound healing, which are independent angiogenesis. At the cellular level, it is most likely that autophagy in endothelial cells is dispensable for their proliferation and migration; however, it is required for their paracrine regulation of the acute inflammatory responses as well as

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A

Atg7EC-/CD31 positive cells (%)

Atg7F/F

CD31

Cell proliferation (OD 450 nm)

EC-/ECC-//-C-/Atg7 At g7EC F/F F /F Atg7 At g7F/

Wound area (μm2)

C

B

20 15 10 5 0

Atg7F/F

300000

Atg7EC-/-

250000 200000 150000 100000 50000 0

0

12

24

(h)

Atg7F/F

Time (day)

ns

25

Atg7EC-/-

200 μm

0h

12 h

24 h

Fig. 4 e The effect of Atg7 endothelial cellespecific knockout on angiogenesis in wounded skin. (A) Left panel: representative CD31 staining in the wounded skin 10 d after injury. Red arrows indicate CD31-positive cells. Scale bars are 30 mm. Right panel: quantified percentage of CD31-positive cells. n [ 3 for each group. ns, nonsignificant. (B) Growth curve of myocardial endothelial cells isolated from Atg7F/F and Atg7ECL/L mice. n [ 6. (C) Migration of myocardial endothelial cells isolated from Atg7F/F and Atg7ECL/L mice. Upper panel: quantified wound areas. n [ 3. Lower panel: representative images of scratched wound healing. (Color version of figure is available online.)

dermal fibroblast and keratinocyte growth, which are essential for skin wound healing. It has been firmly established that two major roles are attributed to endothelial cells during skin wound healing: first, endothelium mediates and regulates the recruitment of leukocytes from the intraluminal compartment to wounded tissues; and second, endothelial cells form new vessels during skin wound repair, which arise from the preexisting microvasculature, a process designated as angiogenesis.24,27 It has been reported that ECs not only function as a transport device for the recruitment of immune cells and regulate leukocyte extravasation at places of inflammation but also have

essential paracrine function by secreting chemokines, interleukins, interferons, and growth factors.28 In this regard, our findings indicate that endothelial cells not only control the recruitment of inflammatory cells but also facilitate epidermal growth for skin wound healing, which are under a negative control by autophagy. Although our results help clarify the controversial observations regarding the role of autophagy in skin wound healing,11-13 they raise several questions regarding the role of autophagy in the control of endothelial celleoriginated paracrine regulation of skin wound healing. First, the acute inflammatory responses including the recruitment of inflammatory cells at the early stage of skin

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Fig. 5 e The effect of Atg7 endothelial cellespecific knockout on the tube formation in vitro and VEGF, bFGF, EGF concentration in ECs supernatants. (A) The effects of Atg7F/F and Atg7ECL/L ECs on the tube formation in vitro. Left panel: Representative pictures of ECs tubular networks after 12 h. Right panel: Quantified total tube length of endothelial cell tube formation. n [ 3 for each group. ns, nonsignificant. Scale bars are 100 mm. (B) The culture supernatant from Atg7F/F and Atg7ECL/L ECs were collected 48 h after incubation, and VEGF, bFGF, and EGF levels were measured by ELISA. n [ 3 for each group. *P < 0.05. (Color version of figure is available online.)

Atg7EC-/MAC2 positive cells (%)

Atg7F/F

Mac2

B

CD3

Atg7F/F

60

**

40 20 0

Atg7EC-/CD3 positive cells (%)

A

3

**

2 1 0

Fig. 6 e The effect of Atg7 endothelial cellespecific knockout on infiltration of inflammatory cells in wounded skin. (A) Left panel: representative Mac2 and CD3 staining in the wounded skin 3 d after injury. Red arrows indicate Mac2-positive cells. Scale bars are 30 mm. Right panel: quantified percentage of Mac2-positive cells. (B) Left panel: representative CD3 staining in the wounded skin 3 d after injury. Red arrows indicate CD3-positive cells. Scale bars are 30 mm. Right panel: quantified percentage of CD3 positive cells. n [ 3 for each group. ns, nonsignificant. (Color version of figure is available online.)

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Cell proliferation (OD 450 nm)

2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0

CM %

Atg7F/F Atg7EC-/-

*

*

50

66.6

Cell proliferation (OD 450 nm)

A

CM % 0

33.3

Atg7F/F Atg7EC-/-

0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0

33.3

Wound area (μm2)

B

Atg7F/F Atg7EC-/-

16000

*

12000

*

8000 4000

12h

24 (h)

50

66.6

*

12000

*

8000 4000 0 0

12

*

Atg7F/F Atg7EC-/-

16000

0 0

0h

Wound area (μm2)

B

*

0h

12 12h

24 (h) 24h

24h Atg7EC-/30μm

Atg7EC-/30μm

Atg7F/F

Atg7F/F

Fig. 7 e The effect of Atg7 endothelial cellespecific knockout on the proliferation and migration of dermal fibroblasts. (A) The effects of conditional medium (CM) from Atg7F/F and Atg7ECL/L ECs on the proliferation of dermal fibroblasts isolated from WT mice. n [ 6. (B) The effects of CM from Atg7F/F and Atg7ECL/L ECs on the migration of dermal fibroblasts isolated from WT mice. Cells were treated with CM with the final concentrations of 50% as indicated. Upper panel: quantified wound areas. n [ 3. Lower panel: representative images of scratched wound healing. *P < 0.05. (Color version of figure is available online.)

wound healing are the driving forces to initiate and regulate the wound healing, and the phenotypic modulation of inflammatory cells such as T lymphocytes and macrophages appear to be crucial for orchestrating the adaptive inflammatory responses for skin wound repair.29e32 We found that the increased infiltration of T lymphocytes and macrophages in the wounded skin is associated with improved skin wound healing in endothelial cellespecific autophagy deficient mice. Hence, it is intriguing whether autophagy-dependent

Fig. 8 e The effect of Atg7 endothelial cellespecific knockout on the proliferation and migration of keratinocytes. (A) The effects of conditional medium (CM) from Atg7F/F and Atg7ECL/L ECs on the proliferation of keratinocytes isolated from WT mice. n [ 6. (B) The effects of CM from Atg7F/F and Atg7ECL/L ECs on the migration of keratinocytes isolated from WT mice. Cells were treated with CM with the final concentrations of 50% as indicated. Upper panel: quantified wound areas. n [ 3. Lower panel: representative images of scratched wound healing. *P < 0.05. (Color version of figure is available online.)

endothelial cell paracrine promotes proinflammatory phenotypes of T lymphocytes and macrophages in skin wound healing. Second, the adaptive inflammatory responses are also linked to appropriate extracellular matrix production and proliferation of dermal fibroblasts and keratinocytes for skin wound healing.29e32 Although we found that autophagy-dependent suppression of endothelial cell paracrineemediated growth of dermal fibroblasts and keratinocytes, whether the autophagy-dependent endothelial cell paracrine regulation of inflammatory responses contributes to the growth of keratinocytes and fibroblasts remains to be determined. Third, the substances derived from endothelial

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j o u r n a l o f s u r g i c a l r e s e a r c h  m a y 2 0 2 0 ( 2 4 9 ) 1 4 5 e1 5 5

cells which are responsible for the autophagy-dependent regulation of skin wound healing remain unknown. In summary, our findings show that the conditional medium of cultured Atg7EC
/
endothelial cells significantly enhanced the proliferation and migration of dermal fibroblasts and keratinocytes in vitro. Deletion of autophagy gene Atg7 of endothelial cell can increase infiltration of T lymphocytes and macrophages in wounded skin is associated with improved skin wound healing, but hardly affect endothelial cell growth and migration thus having minimal impact on angiogenesis in vivo. These results indicate that autophagy deficiency in endothelial cells facilitates their paracrine regulation of the acute inflammatory responses as well as the growth of dermal fibroblasts and keratinocytes thereby enhancing skin wound healing. Finally, the molecular mechanism by which autophagy regulates endothelial cellemediated paracrine regulation of skin wound healing has not been delineated in the present study. Further investigations to clarify these issues may provide novel insights into the understanding of autophagy-mediated endothelial cell paracrine regulation of skin wound healing, thus facilitating the development of a new therapeutic strategy for skin wound repair.

Acknowledgment This work was supported by National 973 Project Grant (2014CB542401), National Nature and Science Foundation of P. R. China (No. 81370267, 81372065, and 81871563), the Fundamental Research Funds for the Central Universities, China (21619357). K.-C.L. and C.-H.W. designed the experiments. K.-C.L., C.H.W., J.-J.Z., and C.Q. performed the research. X.-L.W. and X.S.T. analyzed the data. H.-W.L. and T.C. supervised the experimental procedure.

Disclosure The authors have declared no conflicts of interest.

Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.jss.2019.12.004.

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