Angiopoietin‑Like 4 Stimulates STAT3‑Mediated Inos Expression and Enhances Angiogenesis to Accelerate Wound Healing in Diabetic Mice

This document is downloaded from DR‑NTU (https://dr.ntu.edu.sg) Nanyang Technological University, Singapore.

Angiopoietin‑like 4 stimulates STAT3‑mediated iNOS expression and enhances angiogenesis to accelerate wound healing in diabetic mice

Chong, Han Chung; Chan, Jeremy Soon Kiat; Goh, Chi Qin; Gounko, Natalia V.; Luo, Baiwen; Wang, Xiaoling; Foo, Selin; Wong, Marcus Thien Chong; Choong, Cleo Swee Neo; Kersten, Sander; Tan, Nguan Soon

2014

Chong, H. C., Chan, J. S. K., Goh, C. Q., Gounko, N. V., Luo, B., Wang, X., et al. (2014). Angiopoietin‑like 4 stimulates STAT3‑mediated iNOS expression and enhances angiogenesis to accelerate wound healing in diabetic mice. Molecular therapy, 22(9), 1593‑1604. https://hdl.handle.net/10356/106780 https://doi.org/10.1038/mt.2014.102

© 2014 The Author(s). This is the author created version of a work that has been peer reviewed and accepted for publication in Molecular Therapy, published by Nature Publishing Group on behalf of The Author(s). It incorporates referee’s comments but changes resulting from the publishing process, such as copyediting, structural formatting, may not be reflected in this document. The published version is available at: [http://dx.doi.org/10.1038/mt.2014.102].

Downloaded on 02 Oct 2021 09:34:11 SGT Angiopoietin-like 4 stimulates STAT3-mediated iNOS expression and enhances angiogenesis to accelerate wound healing in diabetic mice.

Running title: ANGPTL4 increases nitric oxide level.

Han Chung Chong1, Jeremy Soon Kiat Chan1, Chi Qin Goh1, Natalia V. Gounko2, Baiwen Luo3, Xiaoling Wang1, Selin Foo1, Marcus Thien Chong Wong4, Cleo Choong3, Sander Kersten5 and Nguan Soon Tan1, 2

1School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551. 2Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, A*STAR, Singapore 138673. 3School of Materials Science and Engineering, Nanyang Technological University, Nanyang Avenue, Singapore 639798. 4Tan Tock Seng Hospital, 11 Jalan Tan Tock Seng, Singapore 308433. 5Nutrition, Metabolism and Genomics group, Wageningen Univeristy, Bomenweg 2, Wangeningen, the Netherlands.

Address correspondence to: Nguan Soon Tan, PhD School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551. Email: [email protected] or [email protected]. Tel: +65 6316 2941; Fax: +65 6791 3856

The authors declare no conflicts of interest for this study.

Abstract

Impaired wound healing is a major source of morbidity in diabetic patients. Poor outcome has, in part, been related to increased inflammation, poor angiogenesis, and deficiencies in extracellular matrix components. Despite the enormous impact of these chronic wounds, effective therapies are lacking. Here we showed that the topical application of recombinant matricellular protein ANGPTL4 accelerated wound re-epithelialisation in diabetic mice, in part, by improving angiogenesis. ANGPTL4 expression is markedly elevated upon normal wound injury. In contrast, ANGPTL4 expression remains low throughout the healing period in diabetic wounds. Exogenous ANGPTL4 modulated several regulatory networks involved in cell migration, angiogenesis and inflammation, as evidenced by an altered gene expression signature. ANGPTL4 influenced the expression profile of endothelial-specific CD31 in diabetic wounds, returning its profile to that observed in wild-type wounds. We showed

ANGPTL4 induced nitric oxide production through an integrin/JAK/STAT3-mediated upregulation of iNOS expression in wound epithelia, thus revealing a hitherto unknown mechanism by which ANGPTL4 regulated angiogenesis via keratinocyte-to-endothelial-cell communication. These data show that the replacement of ANGPTL4 may be an effective adjunctive or new therapeutic avenue for treating poor healing wounds. The present finding also confirms that therapeutic angiogenesis remains an attractive treatment modality for diabetic wound healing.

Keywords: wound healing; angiogenesis; angiopoietin-like 4; inducible nitric oxide synthase

Introduction

Impaired wound repair is a major complication of diabetes, resulting in substantial morbidity, lost productivity, and healthcare expenditures. Furthermore, a poor healing diabetic wound is an open portal for infections, often resulting in chronic inflammation, sepsis, dehiscence, and death. Despite the enormous impact of these chronic wounds, effective therapies remain lacking. Effective management of these problems will require better understanding of the healing process to allow the creation of a salubrious physical and biochemical environment conducive to healing1.

Normal wound healing proceeds via a continuum of events, including phases of acute inflammation, proliferation, and maturation2. The diabetic state induces alterations of all three of these physiological processes3. Diabetic wounds are characterized by accumulation of devitalized tissue, increased/prolonged inflammation, poor wound-related angiogenesis, and deficiencies in extracellular matrix (ECM) components4. Diabetic wounds show elevated levels of matrix metalloproteinases and increased proteolytic degradation of ECM components, culminating in a corrupt microenvironment that cannot support healing3,5. Cells such as keratinocytes, fibroblasts, and endothelial cells also display dysregulated expressions of and responses to many growth factors and cytokines.

These features typically make diabetic wounds poorly responsive to most treatments, and thus it may be most advantageous to intervene with aggressive strategies that could improve the corrupt extracellular microenvironment. In this respect, the role of matricellular proteins in wound healing is of interest. Matricellular proteins can associate with the diverse proteins of the ECM reservoir, bridging them with cognate cell surface receptors. They reside at the crossroads of cell–matrix communication, modulating several regulatory networks6.

Presumably, the regulatory pathways consist of complex networks, creating many

3 opportunities for the compensatory adjustments required for wound repair. Hence, it may be more efficacious to target or replace the necessary matricellular proteins than individual cytokine-mediated candidates.

Angiopoietin-like 4 (ANGPTL4) is a recently identified matricellular protein that is implicated in wound healing7,8. It undergoes proteolytic cleavage after secretion, releasing the

N-terminal coiled-coil domain (nANGPTL4) and a C-terminal fibrinogen-like domain

(cANGPTL4). nANGPTL4 modulates the activity of lipoprotein lipase9. We previously showed that cANGPTL4 facilitates keratinocyte migration and wound re-epithelialisation.

Wounds in ANGPTL4-knockout mice exhibit delayed healing and share many characteristics of diabetic wounds, including reduced expression of matrix proteins, increased inflammation, and impaired wound-related angiogenesis7,8. ANGPTL4 reportedly also has a context- dependent role in angiogenesis and vascular permeability10,11. However, the expression and role of ANGPTL4 in diabetic wound healing remains unclear, and the mechanism by which

ANGPTL4 regulates wound angiogenesis is unknown. The present study investigated the regulation of wound angiogenesis by ANGPTL4 and examined the potential impact of

ANGPTL4 on diabetic wound healing.

Results

Reduced ANGPTL4 expression in impaired diabetic wound healing.

First, we characterized the spatiotemporal expression profile of ANGPTL4 mRNA and

protein during the healing of full-thickness excisional splint wounds in ob/+ (normal) and ob/ob (diabetic) mice. Macroscopic observation on days 7–10 post-injury revealed that wounds of ob/ob mice were closed by 40%, while those of ob/+ mice were completely closed

(Figure 1a and S1a). Haematoxylin and eosin staining of day-3, -5, -7, and -10 post-wound sections showed impaired epithelial regeneration and formation of granulation tissue in ob/ob compared with ob/+ mice. Histomorphometric analysis of centrally dissected ob/ob wound sections revealed significantly delayed re-epithelialisation between days 3–10 post-wounding

(Figure 1b). On day 10 post-injury, the epidermal wound area above the wound bed in ob/ob mice remained larger than in ob/+ mice (Figure S1b). Quantitative real-time PCR (qPCR) revealed reduced ANGPTL4 mRNA levels at day 3 post-wounding in ob/ob compared with ob/+ mice (Figure 1c). Consistently, immunoblot analysis showed a ~5-fold reduction in cANGPTL4 protein level at day 5 post-wounding (Figure 1d), and immunofluorescence staining of ANGPTL4 revealed lower signals in wound epithelia and wound bed in ob/ob compared with ob/+ wounds (Figure S1c). Similar observations were also obtained from wounds of alloxan-induced diabetic mice (data not shown). Antibody specificity was determined by infrared western blot and immunostaining analysis using skin biopsies from wild-type (ANGPTL4+/+) and ANGPTL4-knockout (ANGPTL4−/−) mice (Figure S2a and

S2b).

Topical application of cANGPTL4 improves the healing rate of diabetic wounds.

Next, we examined the effect of a single topical application of recombinant cANGPTL4 on

the diabetic wound healing rate. We inflicted two full-thickness excisional splint wounds on

the dorsal skin of ob/ob mice: one to be treated with saline and the other with cANGPTL4

(Figure S3a). Wound images at days 7 and 10 post-injury showed improved wound closure with cANGPTL4 treatment compared with saline treatment (Figure S3b). Histomorphological examination of wound sections showed that cANGPTL4 significantly accelerated re- epithelialisation compared with saline controls, as indicated by the reduced epithelial gap

(Figure 2a and S3c). Similarly, the length of neo-epithelia from human skin biopsies was significantly dose-dependently increased in the diabetic-like condition with cANGPTL4 treatment compared to in normal or diabetic conditions without cANGPTL4 (Figures 2b and

S3d).

Aberrant growth factor production and activation of cognate signalling cascades contribute to poor diabetic wound healing. To investigate if treatment of diabetic wounds with cANGPTL4 affected their expression profiles, we performed focused qPCR arrays on wound biopsies from control ob/+, and saline- and cANGPTL4-treated ob/ob wounds. We analysed 77 genes clustered according to the biological functions of proliferation, angiogenesis, cell migration, ECM matrix, cell apoptosis, and inflammation. Heat maps revealed significant expression changes in many genes associated with angiogenesis, ECM remodelling, and inflammation (Figure 2c; Table S2, S3, and S4). cANGPTL4-treated ob/ob

wounds exhibited a 61.1% change in overall differential gene profile compared to saline-

treated ob/ob wounds, suggesting an altered wound healing signature (Figure 2d).

ANGPTL4 induces angiogenesis in diabetic wounds.

Next, we examined expressions of markers of specific cell types, including endothelial cells (CD31), myofibroblasts (αSMA), proliferating cells (PCNA), and macrophages (F4/80). Saline-treated ob/ob wounds displayed reduced CD31 and αSMA expressions from day 3 post-wounding compared with ob/+ wounds. F4/80 and PCNA

expressions were elevated until day 10 post-wounding in saline-treated ob/ob wounds

compared with ob/+ wounds, consistent with prolonged inflammation and delayed resolution

of wound re-epithelialisation (Figures 3a and b). Although the F4/80 and PCNA expression pattern remained unchanged in cANGPTL4-treated ob/ob wounds, the overall expression levels were significantly reduced compared to in saline-treated ob/ob wounds (Figure 3b).

Notably, the CD31 and αSMA expression profiles in cANGPTL4-treated ob/ob wounds had reverted to those observed in ob/+ wounds (Figure 3b). To further strengthen our finding, immunofluorescence staining of Ki67, αSMA and CD31 were performed on day 5 and day7 post-wounding biopsies from ob/+, saline- and cANGPTL4-treated ob/ob mice. No difference was observed in number of Ki67-positive proliferating cells among ob/+, saline- and cANGPTL4-treated ob/ob wounds (Figure S4). Consistent with the finding from western blots, immunostaining of CD31 and αSMA in cANGPTL4-treated ob/ob wounds have reverted to those observed in ob/+ wounds (Figure S4).

To determine if ANGPTL4 improves wound angiogenesis, we performed immunofluorescence staining of CD31 and quantitative analysis of CD31-positive isolated cells from various treated day-5 wound biopsies. The Chalkley counting method was applied for analysing the extent of endothelial tube formation wherein a higher Chalkley count represents better angiogenesis. Chalkley counting method is a morphometric point counting systems using microscope eyepiece graticule and has been recommended as a standard in international consensus reports on the quantification of angiogenesis12,13. cANGPTL4 treatment increased CD31 immunohistochemistry staining in ob/ob wounds compared with saline-treated ob/ob wounds (Figure 4a, top), indicating increased wound vasculature. The mean Chalkley counts of the cANGPTL4–treated ob/ob wounds was significantly 2-fold higher compared to saline-treated ob/ob wounds (Figure 4a, bottom). Our FACS analysis showed approximately 53% increase in CD31+ endothelial cells in cANGPTL4-treated ob/ob

wounds compared with saline-treated ob/ob wounds, approaching to the level detected in

ob/+ wounds (Figure 4b). Furthermore, 72.9% of the CD31+ endothelial cell population in

cANGPTL4-treated ob/ob wounds also stained positive for proliferation marker Ki67, compared with only 30.7% and 52.4% (CD31+Ki67+) for saline-treated ob/ob and ob/+ wounds, respectively. Altogether, these observations indicate that cANGPTL4 improves wound angiogenesis.

To check if ANGPTL4 have any direct effect on the endothelial cells (ECs) survival, we have performed FACS analysis for both apoptosis (annexin and PI staining) and proliferation (incorporation of BrdU) assay on attached ECs in the presence and absence of cANGPTL4. Our results did not show any significant difference on apoptosis and proliferation of cANGPTL4-treated ECs compared with saline-treated ECs (Figure S5)

ANGPTL4 increases nitric oxide (NO) production as an angiogenic mediator in diabetic wounds.

Our focused gene array analysis revealed that iNOS expression was dramatically increased in ob/ob wounds treated with cANGPTL4 compared with saline. To understand how ANGPTL4 modulates angiogenesis, we first examined the level of nitric oxide (NO), which is a potent angiogenesis mediator in various time points of our wound biopsies. Using a DAF-FM diacetate-based assay, we detected an overall NO reduction in ob/ob compared with ob/+ wounds (Figure 5a). Furthermore, cANGPTL4-treated ob/ob wounds showed significantly increased NO production from day 3 post-wounding compared to those with saline treatment, suggesting that cANGPTL4 may mediate NO production (Figure 5a).

We further found that, in contrast to ob/+ wounds in which eNOS expression peaked at day 5 post-wounding, eNOS expression in ob/ob wounds prematurely and transiently peaked at day 3 post-wounding (Figure 5b). cANGPTL4 treatment of ob/ob wounds resulted

in an increase in eNOS expression from day 5 post-wounding, more closely resembling the

expression pattern of ob/+ wounds. The maximal fold-change in eNOS expression was

similar between ob/+ and cANGPTL4 treated ob/ob wounds. These observations suggest that

the elevation of eNOS in wounds may be a secondary effect. cANGPTL4 also did not

modulate eNOS expression in primary human dermal microvascular endothelial cells (Figure

5c). Our results showed that ob/ob wounds expressed little iNOS mRNA compared to normal

ob/+ wounds, in which iNOS expression peaked at day 3 post-injury. cANGPTL4 treatment

in ob/ob wounds increased iNOS expression, which peaked at day 7 post-injury (Figure 5d).

Consistently, our immunofluorescence staining for iNOS showed increased intensity in day-7

cANGPTL4-treated wounds compared with saline controls (Figure 5e). To further strengthen our findings, we examined the NO level of ANGPTL4-knockout (KO) mice. Post wounding biopsies from KO mice have significantly lower level of NO detected in the wound epithelium by DAF staining when compared to wild type (WT) mice (Figure 5f). In addition, we separately examined the mRNA expression of iNOS in laser capture microdissected wound epithelia and dermis from the day 7 post-wound biopsies of ob/ob mice (Figure 5g and S6). The iNOS mRNA level in the epithelia of cANGPTL4-treated wounds was 10-fold higher than in saline-treated controls. cANGPTL4 treatment of ob/ob wounds did not affect dermal fibroblast iNOS mRNA expression, indicating a cell type-specific response.

To confirm the roles of iNOS and NO induction of angiogenesis and improvement of diabetic wound healing, we co-treated ob/ob wounds with cANGPTL4 and aminoguandine

(AG), a selective iNOS inhibitor. As expected, AG delayed ob/ob wound healing even with

cANGPTL4 application, phenocopying saline treated ob/ob wounds (Figure 6a).

Histomorphometric analysis of saline treated, cANGPTL4 treated and cANGPTL4 plus AG

treated ob/ob wound sections revealed that the percentage of wound closure at day 7 post-

wounding was lower in both saline treated and cANGPTL4 plus AG treated ob/ob wound

when compared to cANGPTL4 treated ob/ob wound (Figure 6b and S7). Altogether, these

observations suggested that ANGPTL4 stimulates the expression of iNOS in the wound

keratinocytes, elevating NO at the wound site to enhance diabetic wound closure rate.

To further strengthen our findings, we have performed in-vitro tube formation assay

using endothelial cells alone or cocultured with keratinocytes in the presence and absence of

cANGPTL4. Aminoguandine (AG), an iNOS inhibitor, was also used in the assay. The tube

formation assays were performed under normal and diabetic conditions. Our observation showed that tubule formation was impaired in diabetic conditions, albeit only slightly. Tube formation was not affected by cANGPTL4, regarding of culture conditions. As expected, AG only has little effect. Notably, our result showed that cANGPTL4 accelerated tubule formation only in the presence of keratinocytes which was inhibited by AG (Figure S8). Thus,

the tubule-promoting effect of cANGPTL4 works via a keratinocyte-endothelial cells interaction. (Figure S8).

ANGPTL4 regulates iNOS expression via STAT3 activation in keratinocytes.

To decipher the underlying mechanism of cANGPTL4-mediated acceleration of diabetic wound closure, we first examined the interaction between exogenous cANGPTL4 and integrins in ob/ob wounds using proximity ligation assay (PLA). cANGPTL4 binds to integrin and subsequently activates downstream mediators such as FAK, Src and ERK to modulate gene expression 7. PLA signals, visualized as red spots, were increased in

cANGPTL4-treated ob/ob wounds compared to saline-treated wounds, suggesting that

cANGPTL4 bound and activated integrin β1 (Figure 7a, left panel and S9a). This was confirmed using PLA for active integrin β1 and active Rac1 interactions, which showed increased PLA signals per cell in cANGPTL4-treated compared with saline-treated ob/ob wounds (Figure 7a, right panel and S9b). PLA performed with single specific antibody served

10 as negative controls (Figure S9c). Immunoprecipitation using anti-cANGPTL4 followed by immunoblot analysis also showed that the activation of downstream mediators such as phosphorylated FAK and Src (Figure 7b).

In silico analysis of mouse iNOS gene promoter revealed a putative STAT binding site. Immunofluorescence staining showed more phosphorylated STAT3-positive wound keratinocytes in cANGPTL4-treated ob/ob wounds when compared with saline treatment

(Figure 7c). Keratin 6 identifies hyperproliferative wound keratinocytes. We also examined the JAK/STAT3 activation in day 5 and 7 post-wound biopsies from ANGPTL4 KO and WT mice. Interestingly, the expression of both phosphorylated STAT3 and JAK1 proteins, i.e. activated proteins, was significantly reduced in KO when compared with WT (Figure 7d). In addition, immunoblotting of phosphorylated STAT3 and JAK1 proteins from laser microdissected wound epithelia of saline- and cANGPTL4-treated ob/ob wounds clearly showed that cANGPTL4 can activate JAK1/STAT3 signalling in the wound keratinocytes

(Figure 7e). As expected, this activation of JAK1/STAT3 signalling was also observed in laser microdissected wound epithelium of normal human skin compared to diabetic human skin (Figure 7e and S6). To determine whether cANGPTL4 regulates iNOS expression via

STAT3 signalling in keratinocytes, we measured iNOS mRNA by qPCR and NO production using DAF-FM in STAT3-knockdown keratinocytes (KerSTAT3) treated with cANGPTL4. Our result showed reduced iNOS mRNA and NO levels in KerSTAT3 compared with KerCTRL

(Figure 7f and 7g).

Finally, in vivo chromatin immunoprecipitation (ChIP) showed that phospho-STAT3 specifically bound to the cognate responsive elements in the promoter of the mouse iNOS gene in ANGPTL4-treated but not saline-treated ob/ob wounds (Figure 7h, left panel). No immunoprecipitation or amplification was seen with pre-immune IgG or with a control sequence upstream of the responsive elements in the promoter iNOS gene (Figure 7h, left

panel). Quantitative ChIP showed ~15-fold enrichment in STAT-binding region (SBE) when

compared with cognate controls (Figure 7h, right panel). Altogether, these observations

indicate that ANGPTL4 stimulates NO generation in wound keratinocytes through direct transcriptional regulation of the iNOS gene by pSTAT3.

Discussion

Diabetic wounds heal poorly, often resulting in ulceration. Relatively few topical medications

are efficacious for ulcer care, and amputation rates remain high in non-healing diabetic

patients. Growing evidence indicates that altered expressions of cytokines and ECM proteins

play significant roles in the delayed healing of diabetic wounds. Experimental evidence

shows that vascular-endothelial growth factor (VEGF), platelet-derived growth factor, and

epidermal growth factor accelerate diabetic wound healing in animal models14-16; however,

successful results require high doses of these growth factors. Furthermore, these therapeutic

strategies ignore the central coordinating role of the matrix proteins in orchestrating and

enhancing the cascade of cellular events in the wound healing response.

Here we showed that the topical application of recombinant cANGPTL4 accelerated

wound re-epithelialisation in diabetic mice by improving wound angiogenesis. The

matricellular protein cANGPTL4 expression has been weakly detected in normal intact skin

and is markedly elevated upon wound injury. In contrast, cANGPTL4 expression remains

low throughout the healing period in diabetic wounds. Topical application of cANGPTL4

modulates several regulatory networks involved in cell migration, angiogenesis and

inflammation, as evidenced by an altered wound healing gene expression signature when

compared with saline-treated diabetic wounds.

Various studies have suggested that the role of ANGPTL4 in angiogenesis may be

context-dependent. Conflicting roles for ANGPTL4 in tumor vascularization have been

reported17,18. In a model of oxygen-induced retinopathy, pathological neovascularization was

strongly inhibited in ANGPTL4-deficient mice19. A recent study showed that ANGPTL4 improves the cerebral vasculature network after brain injury and displays vasculoprotective properties when used as an adjunct to VEGF20. Evidence from ANGPTL4-knockout mice

indicates that ANGPTL4 is pro-angiogenic in normal wound healing7. These observations

suggest a role for ANGPTL4 in angiogenesis, although the underlying mechanism remains

unresolved. Here we showed that ANGPTL4 has a dramatic influence in the expression

profile of endothelia-specific CD31 in diabetic wounds, returning its profile to that observed

in wild-type wounds. cANGPTL4 induces NO production through an integrin/JAK/STAT3-

mediated upregulation of iNOS expression in diabetic skin, thus revealing a hitherto

unknown mechanism by which ANGPTL4 regulates angiogenesis via keratinocyte-to-

endothelial-cell communication (Figure 8). Interestingly, impaired wound angiogenesis was

also observed in mice that lack α3β1 integrin in epidermis, lending support for a keratinocyte-

to-endothelial-cell crosstalk that is dependent on epithelial integrin-mediated signalling21.

Proximity ligation assay and co-immunoprecipitation analysis revealed that exogenous

cANGPTL4 interacted with integrin β1. ANGPTL4-bound integrins activated downstream

mediators such as Rac1 and JAK. We further showed that ANGPTL4 regulates iNOS

expression via STAT3 activation in keratinocytes as evidenced by immunoblot analysis of

microdissected wound epithelia, STAT3-knockdown keratinocytes and in vivo chromatin

immunoprecipitation. VEGF is a potent angiogenic factor that can also modulate NO

generation via eNOS upregulation 22. In contrast to VEGF, cANGPTL4 had little effect on

eNOS expression, but clearly induced iNOS expression. We observed that ANGPTL4

treatment shifted the eNOS expression peak to day 7 post-wounding, which is most likely due to the increase in proliferating endothelial cells (Ki67+CD31+) already detected in the day-5

wounds. Numerous studies have shown that NO can profoundly impact angiogenesis, and we

are only beginning to understand the mechanism for its observed beneficial effect on wound

repair23,24.

Inflammation plays both positive and negative roles in cutaneous repair – the level and length of inflammation can dictate both the healing time and quality of repair25.

Recapitulating the dampened inflammatory responses found in foetal and adult chronic

wounds has proved useful in accelerating repair and reduced scarring26,27. We observed that

expression of the macrophage marker F4/80 was reduced upon treatment with cANGPTL4

compared to saline, suggesting that cANGPTL4 may reduce wound inflammation. The

relationship between ANGPTL4 and inflammation remains unclear. ANGPTL4 has been

implicated in other inflammation-associated pathologies; it plays a protective role against the

severe pro-inflammatory effects of saturated fat, as well as in arthritis (particularly in models

dependent on adaptive immunity) and in atherosclerosis. The underlying mechanism is

unknown and is likely to be highly complex, beyond the scope of this current work.

We have proposed that cANGPTL4 be topically applied to diabetic wounds, which

limits systemic circulation and therefore reduces unspecific effects on other tissues and

organs. The poorly vascularised state of diabetic wounds further restricts the range of effect

of topical cANGPTL4 application. The expression of cANGPTL4 was strongly elevated

during normal wound healing, but was absent in the diabetic condition. Hence, our treatment

seeks to restore physiological levels of cANGPTL4 functions in diabetic wounds using

exogenous recombinant cANGPTL4 proteins. Indeed, the topical application of cANGPTL4

to the wound bed restore expression profile of many crucial wound healing mediators in diabetic wounds closer to that of normal wounds. Our study suggests that the replacement of matricellular protein ANGPTL4 can provide an adjunctive or new therapeutic avenue for treating poor healing wounds such as diabetes-associated ulcers. Furthermore, the present

results confirm that therapeutic angiogenesis remains an attractive treatment modality for diabetic wound healing.

Materials and methods

Reagents. We used the following antibodies: Ki67 (NCL-Ki67p) and keratin 6 (NCL-CK6)

(NovoCastra, Leica Biosystem, Germany); β-tubulin (H-235, sc-9104), PCNA (PC10, sc-56),

αSMA (alpha-SM1, sc-130617), and goat anti-rabbit and anti-mouse IgG-HRP (Santa Cruz

Biotechnology, USA); CD31 (BD Pharmingen, USA); CD68 (FA-11) (Biolegend); F4/80

(AbD Serotec, UK); STAT3 (ab7966) (Abcam, UK); iNOS/NOS II (06-573) (Merck

Millipore, USA); pStat3(Tyr705) (#9145) (Cell Signaling Technology, USA); ID3 (2B11,

MA1-23242) (Thermo Scientific, USA). cANGPTL4 monoclonal antibodies were produced in-house (clone 4A11H5 and 3F4F5). All secondary Alexa Fluor antibodies were from

Molecular probes (USA). We used the DAB peroxidase substrate kit (Vector Laboratories,

UK). Unless mentioned otherwise, all chemicals were from Sigma-Aldrich and molecular biology enzymes from Fermentas. All oligonucleotides were synthesized by Sigma-Proligo.

Expression and purification of recombinant cANGPTL4. The expression and purification of recombinant cANGPTL4 protein was performed in Drosophila S2 cells as described in our previous publication8. Recombinant ANGPTL4 proteins were purified from the conditioned

medium of stable ANGPTL4-expressing S2 cells by preparative isoelectric membrane

electrophoresis.

Animals and wounding experiments. All animal studies were approved and performed in

compliance with the regulations of the Institutional Animal Care and Use Committee of

Nanyang Technological University. The ANGPTL4-KO mice strain was previously

described28. Male mice aged 6-8 weeks were used in this study and were individually caged

during the wounding experiments. Mice were anesthetized prior to surgery by a single

intraperitoneal injection of 80 mg/kg ketamine/10 mg/kg xylazine. Two circular 5-mm full- thickness excisional splint wounds were created, as previously described29. On the day of

surgery (day 1), 50 µL of recombinant cANGPTL4 protein (1 mg/mL) or PBS in 4%

carboxyl methylcellulose (CMC) was topically applied to the wounds and protected with an

occlusive dressing (Tegaderm™; 3M, USA). Mice were euthanized by CO2 inhalation on

days 1, 3, 5, 7, and 10 post-wounding. Wound biopsies were subjected to photographic

imaging, histomorphometric and biological analyses. Wound biopsies were processed and

wound surface area was determined as previously described29,30. Surface wound area at each

time-point was expressed as a percentage of initial wound area at day 1 (100%).

Histomorphometric measurements of sections were performed as previously described7,29.

In vitro diabetic wound assay. Human skin tissue samples were provided by Dr. Marcus

Wong from Plastic, Reconstructive and Aesthetic Surgery of Tan Tock Seng Hospital,

Singapore under an ethical committee review board approval with reference no. (NHG DSRB

Ref: 2012/00071). All human samples had been de-identified Subcutaneous adipose was removed. Skin biopsies were made using a 4-mm biopsy punch, and were cultured in DMEM supplemented with 10% foetal bovine serum containing either 5.5 mM glucose and 1 nM insulin (normal condition) or 25 mM glucose and 10 nM insulin (diabetic condition) in a

31 humidified incubator with 5% CO2 at 37°C .

Assessment of wound healing. Wounds were photographed at days 1, 3, 5, 7, and 10 post-

wounding, using a Canon G12 digital camera. Each image included a ruler to allow standard

calibration of measurements. Surface wound area was quantified at each time-point, using

Image-Pro® Plus version 5.1.0.20 software (Media Cybernetics, USA), and was expressed as

a percentage of initial wound area at day 1 (100%). Histomorphometric measurements of

sections were made through the centre of the wound. Sections of wound biopsies were

stained with haematoxylin-eosin. Histological images were visualized with Nikon Eclipse 90i

brightfield microscope using a Plan Fluor, 10×/0.30 objective and QCapture Pro version

5.0.1.26 software (QImaging, Canada). The measurements were performed three times from

random sections using Adobe Photoshop CS5.1, and the image pixels were calibrated to µm

using the scale bar.

Laser capture microdissection (LCM). Paraffin-embedded sections were mounted onto

MembraneSlides 1.0 PEN (Carl Zeiss) that was pre-treated with UV for 30 min.

Hematoxylin- and eosin-stained sections were then subjected to LCM using Arcturus® XT laser capture microdissection system according to the manufacturer’s instructions (Life techonologies). LCM tissues were collected onto an adhesive CapSure® Macro LCM Caps

(Life techonologies).

Immunoblot and western blot analysis for LCM samples. The LCM caps with the captured samples were capped onto 500 µL centrifuging tubes containing 15 µL of lysis buffer and process as previously described32. Total RNA was isolated using RecoverAllTM

total nucleic acid isolation (Ambion) according to the manufacturer protocol. Fifty ng of

RNA was subjected to Full SpectrumTM Complete Transcriptome RNA Amplification

(System Biosciences) according to manufacturer’s recommendation prior qPCR.

Immunohistochemistry for CD31. Sections were fixed, processed, and blocked as for

immunofluorescence staining. Sections were incubated overnight at 4°C with rat anti-mouse

CD31 antibody, followed by incubation with biotinlyated goat anti-rat IgG antibody (Vector laboratories, USA). After incubation with secondary antibodies, sections were incubated in avidin:biotinylated enzyme complex (ABC; Vector Laboratories, USA) for 30 min. Sections were developed with 3,3′ -diaminobenzidine (DAB) substrate (Vector Laboratories, USA), which produced a brown stain. Slides were mounted with Fluka Eukitt® quick-hardening mounting medium (Sigma-Aldrich, USA). Negative controls did not include the primary antibody. Images were taken using Carl Zeiss MIRAX MIDI using a Plan-Apochromat

20×/0.8 NA objective and Marlin F146.C camera, and MIRAX Viewer Version 1.11.49.0 software. Chalkley counting procedure was used for the quantification of capillaries in the wound bed as previously described12,13,33,34. Briefly, the three most vascular areas (hot spots)

with the highest number of microvessel profiles were chosen subjectively from each wound

biopsies. A 25-point Chalkley grid was applied to each area and oriented to permit the

maximum number of “hit on” points. The mean Chalkley count was determined.

Chromatin immunoprecipitation (ChIP). Briefly, biopsies from saline- and cANGPT4- treated ob/ob wounds at day 7 post injury were cut into small pieces and then digested with

0.5% collagenase I at 37°C. Cells were retrieved and cross-linked using 0.5% formaldehyde for 10 min at 37°C, followed by sonication in SDS lysis buffer (1% SDS, 10 mM EDTA, and

50 mM Tris-HCl, pH 8.1) to obtain cross-linked DNA of 200–500 bp in length. Of the supernatant, 10% was used as input, while the remaining amount was used to for complexes with pSTAT3(Y705) specific antibody (Cell Signaling Technology, USA) or pre-immune

IgG. Complexes were pulled down by Protein A/G (Santa Cruz, USA) and were eluted with elution buffer (1% SDS, 0.1 M NaHCO3). DNA fragments were reverse cross-linked at 65°C for 6 hr. PCR was performed with 5–10 µL of the eluate, using primers flanking either the

STAT-binding site of the iNOS gene (−942 to −934) or an unrelated control sequence (>2 kbp away from the binding site). The primer pairs used are presented in Table S1.

Immunoblotting. Wound biopsies were homogenized with ice-cold protein lysis buffer (20 mM Na2H2PO4, 250 mM NaCl, 1% Triton X-100, 0.1% SDS, and 1 mM PMSF; pH 8.0).

Total protein lysates were resolved using 10% SDS-PAGE and electrotransferred onto

Immobilon-FL polyvinylidene fluoride membrane (IPFL00010, Merck Millipore, USA).

Membranes were blocked with 1× Odyssey Blocking buffer (#927-40000, LI-COR

Biotechnology, USA) for 1 h at room temperature. The membrane was then incubated

overnight at 4°C with the indicated primary antibodies in 0.5x Odyssey Blocking buffer

diluted with TBST (50 mM Tris HCl, pH 7.6, 150 mM NaCl, and 0.05% Tween-20).

Membranes were washed thrice with TBST, and incubated with appropriate IRDye®680- or

800-conjugated anti-IgG secondary antibodies (1:10000, LI-COR Biotechnology, USA) for 1 h at room temperature. Protein bands were revealed using the Odyssey® CLx Infrared

Imaging System, and signals were quantified using ImageStudio Software (LI-COR

Biotechnology, USA). To detect cANGPTL4, the membrane was blocked overnight at 4°C

with 1× Odyssey Blocking buffer. The membrane was then incubated overnight at 4°C with

anti-cANGPTL4 mouse monoclonal antibodies [clone 4A11H5] at 0.6 µg/cm2 of the

membrane in 0.5× Odyssey Blocking buffer diluted with TBST.

RNA extraction and reverse transcription. Total RNA was extracted using TRIzol®

Reagent followed by the PureLink™ Micro-to-Midi Total RNA Purification System

(Invitrogen, USA) according to the manufacturer’s protocol. Total RNA was quantified based

on the A260/A280 absorbance using the Nanodrop ND1000 (Thermo scientific, USA). Total

RNA was reversed transcribed using iScript Reverse Transcription Supermix for RT-1PCR

(#170-8841, Bio-Rad, USA).

Focused real-time PCR array. Real-time PCR arrays were used to analyse the expression of

a focused panel of genes. qPCR was performed with the CFX96™ real-time PCR detection system (Bio-Rad, USA), using KAPATM SYBR qPCR Universal Master Mix

(KAPABiosystems). Melt curve analysis was included to ensure that only one PCR product

was formed. Primers were designed to generate a PCR amplification product of 100–250 bp.

Only primer pairs yielding unique amplification products without primer–dimer formation

were subsequently used for real-time PCR assays. Expression was normalized to that of the

housekeeping gene ribosomal protein L27, which did not change under any of the studied

experimental conditions. The qPCR primer sequences are available in Supplementary Table

S1.

Hydroxyproline assay. Wound biopsies were frozen in liquid nitrogen, and then thoroughly homogenized in distilled water. Aliquots of samples (50 µL) were hydrolysed in 2 N NaOH

at 120°C for 2 h, and then oxidized with chloramine-T reagent (0.0127 g/mL) for 25 min at room temperature. The chromophore was then developed with the addition of p- dimethylaminobenzaldehyde reagent (0.3 g/mL dissolved in methanol/hydrochloric acid solution, 2:1 v/v). The absorbance of the formed reddish-hue complex was measured at 550 nm using a SpectraMax® M2e Multi-Mode Microplate Reader and SoftMax® Pro

Microplate Data Acquisition & Analysis Software (Molecular Devices, USA). Trans-4-

hydroxy-L-proline (0–300 µg/mL) was included as a standard. The value of unknown

hydroxyproline was determined from the standard curve. Weight of wound biopsies was used

for normalisation.

Nitric oxide determination. Intracellular level of NO from wound biopsies were measured

using cell-permeable 4,5-diamino-fluorescein (DAF-FM diacetate) (D23844, Molecular

probes, Invitrogen, USA). Wound biopsies were lysed in Krebs buffer, and incubated with 10

µM DAF-FM diacetate for 30 min at 37°C in darkness. We immediately recorded the

fluorophore signal at 495 nm excitation and 515 nm emission wavelengths using a GloMax

20/20 Luminometer (Promega, USA). Fluorescence was expressed as arbitrary fluorescence

units (AU), and was measured with the same instrument settings for all experiments.

Intracellular staining and Flow cytometry. For determination of proliferative endothelial

cells, intracellular staining for CD31 and Ki67 was carried out on the cells isolated from fresh

wound biopsy. Cells were fixed and permeabilized in 300 µL of cytoperm buffer (0.01%

paraformaldehyde with 0.1% NP40 in PBS) for 20 min on ice and blocked with 10% normal

goat serum in PBS for 30 min at room temperature. Following that the cells were incubated

with staining buffer (3% NGS in PBS) containing the rat anti-CD31 (1:200) and rabbit anti-

Ki67 (1:200) for 30 minutes on ice, followed by incubation with FITC conjugated goat anti-

rat IgG and PE conjugated goat anti-rabbit IgG secondary antibody for another 30 minutes on

ice before flow cytometric analysis. For determination of nitric oxide, intracellular staining

with cell-permeable 4,5-diamino-fluorescein (DAF-FM diacetate) (D23844, Molecular

probes, Invitrogen, USA) was carried out on the transfected keratinocytes. Cells were

incubated with in DMEM alone phenol red free containing 2mM of DAF-FM diacetate for 30 min in a humidified incubator with 5% CO2 at 37°C. Cells were then trypsinized and

resuspended with DMEM supplemented with 10% FBS phenol red free before flow

cytometric analysis. Flow cytometry was carried out using BD Accuri C6 flow cytometer

(BD Biosciences, USA) and data analysis was performed using Flowjo software version 7.6.5

(Tree Star Inc, USA).

Transient suppression of STAT3 in keratinocytes. Keratinocytes were seeded at 5 x 104

cells in 6 well culture plates and were incubated overnight in a humidified incubator with 5%

CO2 at 37°C prior to the transfection assay. The experimental setups include untreated cells,

non-targeting scrambled siRNA and ON-TARGETplus SMARTpool STAT3 siRNA (L-

003544-00-0005) (Thermo scientific, USA). A final concentration of 5 µM of siRNA and 4

µL DharmaFECT transfection reagent with serum free medium was added to each seeded

well according to the manufacture protocol. Transfection medium was replaced with fresh

media 16 hours after the transfection and analysis were performed within 24 to 96 hour post

transfection.

In vitro endothelial tube formation assay. Keratinocytes was seeded onto the 3.0 µm

transwell inserts with a final density of 4x105 cells/insert. The 24-well plates were coated with 100 μl of Matrigel (Becton Dickinson, Bedford, MA). Primary human umbilical vein endothelial cells (HUVEC) of less than 5 passage were then plated in duplicate at a concentration of 4x104 cells/well in 400 μl of endogrow (endothelial cell growth media)

medium. Tubule formation was monitored by liver imaging.

Statistical analysis. Statistical analyses were performed using two-tailed Mann-Whitney

tests or two-way ANOVA using SPSS v.19 software (IBM Corporation, USA). A p value of

<0.05 was considered significant.

Acknowledgements

We thank the National Medical Research Council, Singapore (IRG10may017) for supporting this research. BL and JSKC are recipients of the Nanyang Technological University and

Nanyang President’s Graduate Scholarships, respectively.

Author contributions: HCC designed, performed, and analysed the experiments in this study, with intellectual input from NST. JSKC, XW and SF performed some animal studies and provided technical help. CQG, NVG, and BL performed electron microscopy. MTCW and

CC contributed patient material for analysis and provided intellectual input. NST provided overall coordination with respect to the conception, design, and supervision of the study.

HCC, JSKC and NST wrote the manuscript with comments from co-authors.

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List of Supplementary Materials

Supplementary Methods and Materials.

Table S1: List of primer pairs sequences.

Table S2. Gene expression in ob/+ wound.

Table S3. Gene expression in Saline-treated ob/ob wound.

Table S4. Gene expression in cANGPTL4-treated ob/ob wound.

Figures S1. Histomorphometric analysis of centrally dissected ob/+ and ob/ob wound sections.

Figure S2. Specificity of monoclonal antibodies against mouse ANGPTL4.

Figure S3. cANGPTL4 improves diabetic wound healing.

Figure S4. Immunofluorescence staining of wound biopsies.

Figure S5.ANGPTL4 has no direct effect on endothelial cells survival and proliferation.

Figure S6. Laser capture microdissection (LCM) of skin specimens.

Figure S7. Aminoguanidine inhibits wound healing by cANGPTL4.

Figure S8. Effect of ANGPTL4 on in vitro endothelial tubule formation.

Figure S9. ANGPTL4 activates the integrin signaling pathway.

Figure Legends

Figure 1. Expression of ANGPTL4 was reduced in diabetic wounds. (a) Photos of normal

(ob/+) and diabetic (ob/ob) wound biopsies taken at days 1, 3, 5, 7, and 10 post-wounding.

Scale bar: 5 mm. (b) Wound closure kinetics of ob/+ and ob/ob mice, with wound surface areas plotted as percentage of day 0 (100%). Data are mean ± SEM, n = 12. ***p < 0.001. (c– d) Relative ANGPTL4 mRNA (c) and protein (d) levels in ob/+ and ob/ob wound biopsies as analysed by qPCR and immunoblotting, respectively. Graph shows the relative cANGPTL4 protein level compared to in the day 0 wound in ob/+ mice. qPCR values were normalized to the housekeeping gene ribosomal protein L27. β-tubulin served as a loading and transfer control for immunoblotting. Data are mean ± SEM, n = 12. *p < 0.05, **p < 0.01, ***p <

0.001.

Figure 2. Topical application of ANGPTL4 improves diabetic wound healing. (a)

Wound closure kinetics of saline- and cANGPTL4-treated ob/ob wounds, with wound surface areas plotted as percentage of day 0 (100%). Data are mean ± SEM, n = 12. *p < 0.05, **p <

0.01. (b) Average lengths of neo-epithelium from human skin biopsies cultured for five days under normal and diabetic conditions with various cANGPTL4 concentrations. Data are mean

± SEM, n = 12. *p < 0.05, **p < 0.01. (c) Heat maps show gene expression profiles of ob/+, and saline- and cANGPTL4-treated ob/ob wounds. Genes are clustered according to the following biological gene functions: proliferation, angiogenesis, migration, ECM, apoptosis, and inflammation. The colour spectrum from blue to red depicts the fold-change in LOG form from −1.0 to 1.0. (d) Venn diagram comparing total number of genes from ob/+ (blue), and saline- (red) and cANGPTL4-treated (black) ob/ob wounds. Comparisons of the percentages of total numbers of genes changed.

Figure 3. ANGPTL4 improves wound-related angiogenesis. (a) Representative immunoblots and (b) graph showing relative protein levels of indicated proteins from ob/+, and saline- and cANGPTL4-treated ob/ob wound biopsies. The graph was determined based on the mean densitometry value of the immunoblot obtained from 3 independent set of mice wounding experiments. The data was normalized against the densitometry value of β-tubulin, which served as a loading and transfer control. Data are mean ± SEM, n = 12 mice per set. *p

< 0.05; **p < 0.01, ***p < 0.001 by unpaired Student’s t-test.

Figure 4. ANGPTL4 induced angiogenesis in ob/ob wound. (a) Representative immunostaining for CD31 (brown) on ob/+, and saline- and cANGPTL4-treated ob/ob wound biopsies. Scale bar: 40 µm. Chalkley’s 25 points grids were used to determine the mean chalkley counts shown in the graph for at least 3 microscopic fields from 3 independent experiments. Data are mean ± SEM, n = 9. ***p<0.001; (b) Representative histograms generated from flow cytometry showing percentage of CD31+ endothelial cells population from total cells isolated from ob/+, saline treated ob/ob and cANGPTL4 treated ob/ob wound biopsies (left panel). Histograms gated from CD31+ endothelial cells population showing percentage of Ki67+ proliferative cells (right panel).

Figure 5. ANGPTL4 regulates NO production in wound epithelium. (a) Nitric oxide production in ob/+, and saline- and cANGPTL4-treated ob/ob at indicated post-wounding days, determined by measuring the relative DAF-FM diacetate fluorescence level (arbitrary unit, AU). Values were normalized to total protein. (b–d) Relative mRNA levels of eNOS (b) and iNOS (d) from indicated wound biopsies, and from primary human dermal microvascular endothelial cells (c), as analysed by qPCR. Values were normalized to the housekeeping gene ribosomal protein L27. (e) Immunofluorescence staining of iNOS (red) in day 7 wound

30 biopsies of saline- and cANGPTL4-treated ob/ob mice. Dotted lines delineate the epidermis– dermis interface, scale bar: 50 µm. WB: wound bed. (f) DAF-FM diacetate fluorescence

(green) staining of day 5 post wounding WT and KO mice. Graph shows the mean fluorescence intensity from at least 3 microscopic fields of 3 independents mice. Value are mean ± SEM, n = 12. ***p < 0.001 compared to WT mice. (g) Expression of iNOS mRNA from laser capture microdissected wound epithelium and wound dermis of saline treated ob/ob mice and cANGPTL4 treated ob/ob mice. mRNA level were normalized to 18s ribosomal RNA. Data are mean ± SEM, n = 3. ***p < 0.001 compared to saline treated ob/ob mice.

Figure 6. ANGPTL4 regulates iNOS expression (a) Photographs of ob/ob wounds treated with either saline, cANGPTL4 alone, or cANGPTL4 and aminoguanidine (AG).

Representative pictures from three independent experiments from are shown. Scale bar: 5 mm. (b) Box and whisker plot of the percentage of wound closure in saline treated, cANGPTL4 treated and cANGPTL4 plus AG treated ob/ob day 7 post wounds biopsies sections. Histomorphometric measurements were determined from 10 independent sections from the various wound biopsies. Middle bar = median, box = interquartile range, whiskers = full value range, * = possible outlier.

Figure 7. ANGPTL4 activates JAK1/STAT3 signalling to regulate iNOS expression in keratinocytes. (a) Quantification of PLA signals of cANGPTL4:active integrin β1 (left panel) and active integrin β1:active Rac1 (right panel). Figure S6 presents representative images of all PLA detections. Each PLA signal indicates one detected interaction event. Number of

PLA signals per nucleus was determined using Blobfinder software. Data are mean ± SEM, n

= 3. *p < 0.05, **p < 0.01. (b) Immunodetection of indicated proteins from anti-cANGPTL4

immunoprecipitates of saline- or cANGPTL4-treated ob/ob wound biopsies. β-tubulin from total tissue lysate was used to verify equal loading. (c) Dual immunofluorescence staining of pSTAT3(Y705) (green) and CK6 (red) in saline- and cANGPTL4-treated ob/ob wounds.

Dotted lines delineate the epidermis–dermis interface. Nuclei are stained with DAPI (blue)

Scale bar: 40 µm. WB: wound bed. (d) immunodetection of pSTAT3(Y705), total STAT3, p-

JAK1 and total JAK1 in post wounding D5 and D7 of both KO and WT mice. (e) immunoblots of pSTAT3(Y705), total STAT3, pJAK1 and total JAK1 from laser capture microdissected epithelium of normal human skin, diabetic human skin, saline treated ob/ob wound and cANGPTL4 treated ob/ob wound. (f) Representative histograms generated from flow cytometry showing the mean intensity of DAF-FM signal from STAT3 knockdown keratinocytes in the absence or presence of cANGPTL4, Data are mean ± SEM, n = 3 (g)

Relative mRNA expression level of iNOS from STAT3 knockdown keratinocytes in the absence or presence of cANGPTL4, n = 3. **p < 0.01, ***p < 0.001. (h) ChIP assays were conducted using pre-immune IgG or antibodies against pSTAT3(Y705) in saline- (U) and

cANGPTL4-treated (T) ob/ob wounds at day 7 post-injury. Graph (right panel) shows

quantitative ChIP as percentage of input. Specific regions spanning promoter binding sites of

the iNOS gene were amplified using appropriate primers (Table S1). A control region served

as a negative control.

Figure 8. Schematic illustration of the autocrine and paracrine effect of ANGPTL4 on

cutaneous wound healing. cANGPTL4 can interact with integrin β1, and activate the Src,

ERK, and AKT signalling cascades, leading to subsequent JAK1/STAT3 activation.

Activated STAT3 can then mediate the upregulation of iNOS expression by specifically binding to the cognate responsive elements in the promoter of iNOS gene. Wound

32 angiogenesis can then be promoted by the increase in NO production from the wound keratinocytes in the wound microenvironment.

Chong et al., Figure 1 a days post-wounding b 1 35710100 ob/+ *** ob/ob normal 80 (ob/+) 60

*** 40 *** *** % of Day 0 20 diabetic 0 (ob/ob) 135710 days post-wounding

** c 0.9 ob/+ d day post ob/+ ob/ob ob/ob *** 0.8 wounding 0 3 5 7 10 035710kDa 0.7 cANGPTL4 35 0.6 β-tubulin 55 0.5 *** 3.0 ob/+ * 0.4 2.5 ob/ob ** 0.3 2.0 -tubulin) (ANGPTL4/L27) 0.2  1.5 ** **

relative mRNA expression 0.1 1.0 0 0.5 0357 day post wounding 0 relative expression level (cANGPTL4/ 035710 day post wounding Chong et al., Figure 2 a c ob/ob + ob/ob + ob/+ saline cANGPTL4 ob/ob 100 035710 035710035710 AKT cANGPTL4 CDK4 CDKN2B 80 * CSTA saline EGFR FAS FGF2 FOSL2 60 GDF3 GMCSF HGF * iNOS 40 JUN-B % of Day 0 *** KGF *** KI67 MAP4K1 20 Proliferation MAPK3 PPARA PPARB SKIL SMAD3 0 SOCS1 STAT1 13 57 10 TGFA TGFB1 days post-wounding TGFBR1 XRCC1 b ADIPOQ

m) ANGPT1 ANGPT2  1400 ** FGF1 iNOS 1200 PDGFA * PDGFB PECAM 1000 SMAD3 * STAT3 TGFBR1 800 VEGFA EPHA3 600 EPHB3 FOSB ITGAV 400 MAP4K1 MAPK3 PPARG 200 RHOA

Migration SMAD3 STAT3 0 TGFBR1 TNFAIP2 ADAM9 g/mL) g/mL) GLI1 Length of neo-epithelium (   MMP13 Normal Diabetic MMP9 OPN PAI1 SPARC Diabetic with TGFB2 Diabetic with ECM Angiogenesis TIMP1 TIMP2 TIMP3 TNC d cANGPTL4 (6 TSP-1 cANGPTL4 (12 IAP2 IGFBP4 STAT1 ob/+ CCL11 CCL2 ob/ob + saline CXCL1 CXCL10 ob/ob + cANGPTL4 CXCL5 CXCL9 HIF1A IL-10 IL-18 IL1R1 IL-6 INHBA iNOS KLF9 39 38 SOCS3

42 35 Inflammation Apoptosis 30 47 STAT1 STAT5A TLR2 TLR4 TNFA 49.4% 46.0% VHL 61.1% -1.0 1.0 Chong et al., Figure 3 ob/ob + ob/ob + a days post ob/+ saline cANGPTL4 wounding 035710 035710 035710kDa CD31 130 0.3 1.0 0.6 0.4 0.3 0.2 0.1 0.2 0.2 0.1 0.1 0.6 0.5 0.4 0.4 -SMA 42 1.7 3.1 1.7 1.8 1.6 1.6 0.5 0.6 0.8 0.4 1.9 2.3 1.5 1.4 1.4 F4/80 160 0.1 0.7 0.3 0.2 0.2 0.1 0.5 1.0 1.2 0.6 0.1 0.3 0.5 0.5 0.3 PCNA 35 0.2 1.2 1.0 1.1 0.2 0.2 1.0 1.6 1.0 0.7 0.2 0.5 1.2 1.3 0.5 β-tubulin 55 b * *** 1.5 ob/+ 3.5 *** ** ob/ob + saline 3.0 ns ns *** ob/ob + cANGPTL4 *** 2.5 *** *** 1.0 ** *** *** ns -tubulin) * ns -tubulin) 2.0

 ** ** * ns  1.5 ** 0.5 * * 1.0

* SMA/ 0.5  (CD31/ ( relative expression

0 relative expression 0 035710 03 5710 day post wounding day post wounding ** * 1.5 * ** 2.0 * ** *** * * * *** * * ** 1.5 * ns * 1.0 * * -tubulin) -tubulin) * *   ** 1.0 * 0.5 0.5 (F4/80/ (PCNA/ relative expression relative expression 0 0 035710 035710 day post wounding day post wounding Chong et al., Figure 4

a ob/ob + ob/ob + ob/+ saline cANGPTL4

CD31 Chalkley 25 pts grid

ns 10 *** 9 *** 8 7 6 5 4 3 2 Chalkley count (mean) 1 0 ob/+ ob/ob + ob/ob + saline cANGPTL4

b Negative ctrl (0.7%) Negative ctrl (20.9%) ob/+ (4.5%) ob/+ (52.4%) ob/ob + saline (2.6%) ob/ob + saline (30.7%) ob/ob + cANGPTL4 (4.0%) ob/ob + cANGPTL4 (72.9%) 200 40 CD31+ Ki67+ in CD31+ population 150 30

100 20 Counts Counts 50 10

0 0 103 104 105 106 102 103 104 105 106 107 Intensity Intensity Chong et al., Figure 5 a b Nitric oxide (NO) production ob/+ 7 ob/ob + saline ob/ob + cANGPTL4 2.0 ob/+ 6 ** ob/ob + saline 5 1.5 ob/ob + cANGPTL4 * 4 * * ** 1.0 3 *** *** * 2 0.5 *** Relative mRNA 1 ** fluorescence unit (AU) 0.0 0 expression (eNOS/L27) 01 3 5 7 10 Relative DAF-FM Diacetate 0135710 day post wounding day post wounding e c d iNOS DAPI Endothelial cells D7 ob/ob + saline 3.0 2.0 ob/+ ob/ob + saline 2.5 ns 1.5 ob/ob + cANGPTL4 2.0 WB *** 1.5 1.0

)(eNOS/L27) 1.0 * -3 0.5 D7 ob/ob + cANGPTL4 0.5

Relative mRNA *** (10

relative mRNA level relative mRNA *** ** *** 0 0.0 *** cANGPTL4 - + expression (iNOS/L27) 01357 10 day post wounding WB f g D5 DAF-FM Diacetate WT Laser Capture Microdissection (LCM) wound wound 25 epithelium dermis *** 20 1.1 0.9 0.7 15 0.5 *** 0.3 10 0.1 KO D5 (iNOS/18s) 0.004 5 0.003 ns Mean flouresecence Intensity relative mRNA level relative mRNA 0.002 0 WT KO 0.001 0

ob/obsaline + ob/ob + ob/obsaline +ob/ob + cANGPTL4 cANGPTL4 Chong et al., Figure 6

a Day 7 post-injury ob/ob wounds cANGPTL4 + saline cANGPTL4 saline AG

ob/ob saline

ob/ob cANGPTL4 + AG

ob/ob cANGPTL4

010 20 30 40 50 60 70 80 90 100 % of Wound closure at post wounding Day 7 Chong et al., Figure 7 1 1  a  b 2.5 * 2.5 * ob/ob + ob/ob + ** ** saline cANGPTL4 2.0 IP: cANGPTL4 * 2.0 * kDa IB: integrin 1 1.5 88 1.5 IB: p-FAK 125 1.0 1.0 IB: p-Src 60 0.5 0.5 β-tubulin 0 0 55 PLA signals (spots/nucleus) PLA signals (spots/nucleus) ob/+

cANGPTL4 & active integrin ob/+ ob/obsaline + ob/ob + active Rac1 & integrin ob/obsaline + ob/ob +

cANGPTL4 c cANGPTL4 d D5 D7 pSTAT3 DAPI CK6 Day 7 wound biopies WT KO WT KO ob/ob + saline ob/ob + cANGPTL4 kDa p-STAT3 88 (Y705) 1.50.5 0.45 0.1 Total STAT3 88

WB p-JAK1 130 WB 1.00.1 0.2 0.05 Total JAK1 130

e f g Wound epithelium from LCM 200 1.6 human mouse *** 1.4 150 1.2 1.0 DAF-FM+ ** 0.8 Normal Diabetic ob/ob ob/ob + cANGPTL4 kDa 100 p-STAT3 88 0.6

(Y705) Counts 1.8 0.2 0.2 1.5 (iNOS/18s) 0.4 50

Total STAT3 88 level relative mRNA 0.2 0 p-JAK1 130 + CTRL 0 STAT3 1.8 0.7 0.2 0.8  3 4 5 6 7 STAT3 10 10 10 10 10 Ker  Intensity Ker Total JAK1 130 Ker KerCTRL (52,615) cANGPTL4 KerCTRL + cANGPTL4(158,214)

KerSTAT3 + cANGPTL4 (90,901) SBE control 0.07 *** 0.06 anti-pSTAT3 h SBE control IgG 0.05 p-STAT3 p-STAT3 (Y705) IgG (Y705) IgG 0.04 M UTUTUTUT 0.03

IP (% of input) IP 0.02 0.01 0 UT UT Chong et al., Figure 8

cANGPTL4

  Re-epithelization Keratinocytes Migration

Cytoplasm P P P Src

JAK1/2 FAK Nucleus P P iNOS pSTAT3 pSTAT3

Aminoguandine (AG) NO

Endothelial cells Angiogenesis Angiopoietin-like 4 stimulates STAT3-mediated iNOS expression and enhances angiogenesis to accelerate wound healing in diabetic mice.

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Table S2. Gene expression in ob/+ wound

GenBank Accession Symbol Day 0 Day 3 Day 5 Day 7 Day 10 No. PROLIFERATION 10B -81% 10B *') 10B &67$ 10B 7*)% 10B ;5&& 10B .L 10B )26/ )5$ 10B 6.,/ 6QR1 10B $.7 10B +*) 10B 0$3. E 10B &'. 10B 62&6 10B .*) )*) 10B 7*)$ 10B S &'.1% $) 6PDG 10B )*) 10B 67$7 10B 0$3. 10B (*)5 10B 33$5ȕį 10B )$6 10B 7*)%5 $/. 10B L126 126 10B *0&6) 10B 33$5Į ANGIOGENESIS 10B $1*37 10B 9(*)$ %& 67$7 10B 3'*)% $) 6PDG 10B 3'*)$ 10B 7*)%5 $/. 10B $1*37 10B )*) 10B L126 126 10B 3(&$0 10B $GLSRT

MIGRATION 10B (3+$ 10B 0$3. E %& 67$7 10B )26% 10B ,7*$9 $) 6PDG 10B (3+% 10B 0$3. 10B 5+2$ 10B 7*)%5 $/. 10B 71)$,3 10B 33$5Ȗ ECM 10B 7,03 10B 231 6SS 10B 71& 10B 003 10B 003 10B 763 10B $'$0 10B 63$5& 10B 7,03 10B 7,03 10B 3$, 6HUSLQH 10B 7*)% 10B */, APOPTOSIS 10B ,$3 10B 67$7 10B ,*)%3 INFLAMMATION 10B 71)$ 10B &;&/ 10B ,/ 10B &;&/ 10B &;&/ 10B &&/ 10B ,1+%$ 10B 62&6 10B 7/5 10B ,/

%& ,/5 10B +,)$ 10B 7/5 10B &;&/ 10B 67$7$ 10B 9+/ 10B 67$7 10B ,/ 10B ./) 10B &&/ 10B L126 126

Table S3. Gene expression in Saline-treated ob/ob wound

10B 3(&$0 10B $GLSRT MIGRATION 10B (3+$ 10B 0$3. E %& 67$7 10B )26% 10B ,7*$9 $) 6PDG 10B (3+% 10B 0$3. 10B 5+2$ 10B 7*)%5 $/. 10B 71)$,3 10B 33$5Ȗ ECM 10B 7,03 10B 231 6SS 10B 71& 10B 003 10B 003 10B 763 10B $'$0 10B 63$5& 10B 7,03 10B 7,03 10B 3$, 6HUSLQH 10B 7*)% 10B */, APOPTOSIS 10B ,$3 10B 67$7 10B ,*)%3 INFLAMMATION 10B 71)$ 10B &;&/ 10B ,/ 10B &;&/ 10B &;&/ 10B &&/ 10B ,1+%$ 10B 62&6

10B 7/5 10B ,/ %& ,/5 10B +,)$ 10B 7/5 10B &;&/ 10B 67$7$ 10B 9+/ 10B 67$7 10B ,/ 10B ./) 10B &&/ 10B L126 126

Table S4. Gene expression in cANGPTL4-treated ob/ob wound

10B 7/5 10B ,/ %& ,/5 10B +,)$ 10B 7/5 10B &;&/ 10B 67$7$ 10B 9+/ 10B 67$7 10B ,/ 10B ./) 10B &&/ 10B L126 126

Supplementary Figure Legends

Figure S1. Histomorphometric analysis of centrally dissected ob/+ and ob/ob wound sections.+LVWRPRUSKRPHWULFDQDO\VLVRIFHQWUDOO\GLVVHFWHGREDQGREREZRXQGVHFWLRQV

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Figure S2. Specificity of monoclonal antibodies against mouse ANGPTL4.6SHFLILFLW\RI

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Figure S3. cANGPTL4 improves diabetic wound healing. D $ VFKHPDWLF GLDJUDP

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Figure S5. ANGPTL4 has no direct effect on endothelial cells survival and proliferation.

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Figure S7. Aminoguanidine inhibits wound healing by cANGPTL4. D 3KRWRJUDSKVRI

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Figure S9. ANGPTL4 activates the integrin signaling pathway 'HWHFWLRQ RI D

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Chong et al., Figure S1 a days post-wounding 35710 ob/+ WB WB WB WB

ob/ob WB WB

WB WB b epidermal wound area 20 ob/+ ob/ob 16 ** 2 *** m ** *** P

12 5

x 10 8

0 135710 days post-wounding

Chong et al., Figure S2 a

-/- +/+ -/- +/+

D3 ANGPTL4D3 ANGPTL4D5 ANGPTL4D5 ANGPTL4kDa full length 55 anti-ANGPTL4 (4A11H5) truncated 35

E-tubulin 55

b ANGPTL4+/+ ANGPTL4+/+ ANGPTL4-/- (NEG CONTROL)

anti-ANGPTL4 (4A11H5) DAPI WB WB WB

AF AF Chong et al., Figure S3 a b days post-wounding 35710 cANGPTL4 saline

5 mm saline

R L

cANGPTL4

days post-wounding c 35710

saline WB WB WB WB

cANGPTL4 WB WB

WB WB d Diabetic condition with Diabetic condition with Normal condition Diabetic condition 6 Pg/mL of cANGPTL4 12 Pg/mL of cANGPTL4 Chong et al., Figure S4

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ob/+ WB WB WB

D5 D7 D5

ob/ob + saline WB WB WB

D5 D7 D5 ob/ob + cANGPTL4

WB WB WB Chong et al., Figure S5 a saline 6 Pg/ml cANGPTL4 12 Pg/ml cANGPTL4 106 106 106

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G2/M (7.8%) G2/M (6.5%) G2/M (7.3%) 103 103 103 BrdU FITC-A G0/G1 (75.6%) G0/G1 (70.3%) G0/G1 (76.0%) 102 102 102

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Laser Capture Microdissection (LCM) before after

ob/ob + saline

ob/ob + cANGPTL4

normal human skin

diabetic human skin

150Pm Chong et al., Figure S7 a Day 7 post-injury ob/ob wounds cANGPTL4 + saline cANGPTL4 saline AG

b Day 7 post-injury ob/ob wounds

saline

cANGPTL4

cANGPTL4 + AG Chong et al., Figure S8 a Endothelial cells monoculture Saline cANGPTL4 cANGPTL4 + AG

Normal condition

Diabetic condition

b Endothelial cells co-culture with Keratinocytes Saline cANGPTL4 cANGPTL4 + AG

Normal condition

Diabetic condition Chong et al., Figure S9 a cANGPTL4 & active integrin E1 b active Rac1 & active integrin E1 ob/ob + saline ob/ob + saline ob/ob + saline ob/ob + saline

ob/ob + cANGPTL4 ob/ob + cANGPTL4 ob/ob + cANGPTL4 ob/ob + cANGPTL4

ob/+ ob/+ ob/+ ob/+

c negative control negative control

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