Effect of Resveratrol on Modulation of Endothelial Cells and

Article

The effect of resveratrol on modulation of endothelial cells and macrophages for rapid vascular regeneration from electrospun poly(#-caprolactone) scaffolds Zhihong Wang, Yifan Wu, Jianing Wang, Chuangnian Zhang, Hongyu Yan, Meifeng Zhu, Kai Wang, Chen Li, Qingbo Xu, and Deling Kong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 25 May 2017 Downloaded from http://pubs.acs.org on May 26, 2017

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ACS Applied Materials & Interfaces

The effect of resveratrol on modulation of endothelial cells and macrophages for rapid vascular regeneration from electrospun poly(ε-caprolactone) scaffolds Zhihong Wang1,#, Yifan Wu2,#, Jianing Wang2, Chuangnian Zhang1, Hongyu Yan2, Meifeng Zhu2, Kai Wang2,*, Chen Li1, Qingbo Xu3, Deling Kong1,2,* 1

Tianjin Key Laboratory of Biomaterial Research, Institute of Biomedical

Engineering,Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300192, China 2

Key Laboratory of Bioactive Materials of Ministry of Education; State Key

Laboratory of Medicinal Chemical Biology, College of Life Science, Nankai University, Tianjin 300071, China 3

Cardiovascular Division, King's College London BHF Centre, London, United

Kingdom

#: The authors contributed equally to this work

* Corresponding author

Deling Kong, PhD Tel: 0086 22 23502111; E-mail: [email protected]

Kai Wang, PhD E-mail: [email protected]

ACS Paragon Plus Environment


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Abstract: Rapid endothelialization is one key factor that determines the success of small diameter vascular grafts as an artery substitute in the treatment of cardiovascular disease. Aimed to facilitate vascular regeneration, we developed a vascular scaffold loaded with resveratrol, which is a natural compound extracted from plants and showed multifaceted effects in cardiovascular protection. The tubular poly (ε-caprolactone) (PCL) scaffold was prepared by electrospinning with resveratrol in the PCL solution. In vitro assay demonstrated that resveratrol could be released from the scaffolds in a sustained and controlled manner. Cell culture results indicated that the migration of endothelial cell, NO production and the ability of tube formation all increased in the resveratrol-containing PCL scaffold groups compared to the PCL control. Meanwhile, level of TNF-α, the main pro-inflammatory factor, secreted from macrophages was reduced, and messenger RNA expressions of the M2 macrophage-related genes were increased in the resveratrol-containing group. Further, in vivo implantation was performed by replacing rat abdominal aorta. We observed fast

endothelialization

and

enhanced

vascular

regeneration

in

rats

with

resveratrol-containing scaffolds. The presence of resveratrol also induced a large number of M2 macrophages to infiltrate into the graft wall. Taken together, the incorporation of resveratrol into the PCL grafts enhanced vascular regeneration by modulation of endothelial cells and macrophages.

Keywords: resveratrol; vascular grafts; endothelialization; vascular regeneration; macrophage


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Introduction Small diameter vascular grafts (SDVGs) are widely investigated as an artery substitute in cardiovascular disease treatment. However, the challenge remains because of the lack of endothelium on the graft, which caused acute thrombosis and intimal hyperplasia. Many attempts have been made to facilitate endothelialization on the luminal surface of the graft by pre-seeding with cells1 or incorporating growth factors2-3 and genes.4 Despite some improvements by these approaches, some recognized drawbacks remain unresolved, such as complicated preparation process, poor repeatability, low stability and high production cost. Resveratrol is a natural polyphenol, known for its presence in grape skins and red wines. It has been extensively studied for its anti-cancer, anti-aging and anti-oxidant properties. In recent years, scaffolds loaded with resveratrol have been implicated in tissue engineering and drug delivery. The therapeutic potential of scaffolds in bone, cartilage and neural tissue engineering was improved by loading resveratrol. Studies showed that porous PCL scaffold with resveratrol improved mineralization in vitro.5-7 Tuoh Wu et al fabricated an injectable hydrogel containing oxidized hyaluronic acid and resveratrol, and proved it could reverse chondrocyte degeneration by reducing inflammation and chondrocyte damage.8 Jeffrey et al reported that the compliant nanocomposite (NC) doped with resveratrol as anti-oxidant reduced tissue responses.9 Additionally, resveratrol has been a promising drug candidate for the prevention of cardiovascular diseases due to its multifaceted effects on cardiovascular protection.10 It has been reported to possess multiple protective effects on vascular cells.11 Among these cells, its beneficial impact on the endothelium is essential. Meanwhile, the mechanisms of how resveratrol impact endothelial cells (ECs) have been extensively studied.12 Additionally, resveratrol has been proved to increase proliferation and functional activity of endothelial progenitor cells (EPCs).13 Furthermore, the positive impact of resveratrol on rapid re-endothelialization has been shown in rats with intima-injury14 and implanted with vessel grafts.15 However, the aqueous solubility of resveratrol was lower than 1 mg/ml.16 Besides, the bioavailability of resveratrol was only 2% by oral administration, which



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suggests limited in vivo delivery efficiency of resveratrol, especially for those have to reach target tissues. Therefore, it is necessary to investigate a more effective delivery system for in vivo resveratrol administration. Electrospinning is a simple, effective and single-step method to prepare of nanostructured materials. It offers an opportunity for direct encapsulation of different types of drug molecules, including proteins such as bone morphogenetic protein 2 (BMP2), dexamethasone (DEX), transforming growth factor β1 (TGFβ1),17-18 small molecular drugs19 and many other agents.20 Drug loaded-fibers can deliver the drugs to the targeted sites and enable sustained and controlled release of the selected drugs. Electrospinning has also been demonstrated as an effective method to alter the dissolution profile of drugs with poor solubility in water.21 Herein, we have developed a scaffold loaded with resveratrol by electrospinning method to achieve rapid endothelialization. Poly (ε-caprolactone) (PCL) was chosen as the basal scaffold due to its appropriate mechanical properties and biocompatibility. Previous work has demonstrated that it is easily produced into tubular scaffolds containing similar compliance to that of native vessels.22 Besides, it has been widely applied as a drug delivery with advantages in many aspects.23 Subsequently, we assessed the effect of the resveratrol-containing scaffold in vitro using human umbilical vein endothelial cells (HUVECs) for its functions on endothelial cell proliferation, migration and nitric oxide production. Meanwhile, we also studied its effect on the anti-inflammatory activity and polarization ability using RAW264.7 macrophages. We further investigated the feasibility of these resveratrol-containing PCL scaffolds as small diameter vascular grafts by examining their in situ performance in the rat abdominal aortic replacement model. Neo-tissue formation, vascularization and endothelialization were analyzed.

2. Materials and methods 2.1 materials PCL polymer (Mn, 80 kDa) and resveratrol were purchased from Sigma (USA). Methanol and chloroform were provided by Tianjin Chemical Reagent Company


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(Tianjin, China). Animals were obtained from the Laboratory Animal Center of the Academy of Military Medical Sciences (Beijing, China).

2.2 Preparation of Scaffolds Scaffolds were prepared by electrospinning PCL solution with resveratrol. The weight ratio of resveratrol and PCL were 0.25%, 0.5% and 1.0%. And the fabricated scaffolds were named as PCL-0.25%res, PCL-0.5%res and PCL-1.0%res, respectively. The fabrication process was performed following our previous works.24-25 Briefly, the electrospinning solutions were prepared by dissolving the PCL and resveratrol in methylene chloride and methanol (5:1, v/v) mixture. The final concentration of PCL was 25% (w/v). Mesh scaffolds was fabricated using a grounded metal as collector, whereas a stainless steel rod collector (OD = 2 mm) for tubular grafts. The electrospinning was performed with a flow rate 8 ml/h and a voltage 11 kV with a 21-G needle. The needle tip-collector distance was 17 cm for mesh scaffolds and 11 cm for tubular scaffolds. To sufficiently remove the residual solvents, these scaffolds were treated under vacuum at least 3 days. Scaffolds were sterilized by ethylene oxide for the cell assays and implantation experiment.

2.3 Characterization of scaffolds The fiber morphology of the scaffolds was observed under a scanning electron microscopy (SEM). The average fiber diameter and pore size were analyzed using ImageJ 1.48v software (NIH). Mechanical properties of scaffolds were assessed by a tensile-testing machine with a load capacity of 1KN (Intron). Samples with 1 cm width, 350 µm thickness and 3 cm length in each scaffold group (n=5) were prepared. The inter-clamp distance was set as 1 cm and then samples were pulled longitudinally at a rate of 10 mm/min until rupture. The stress-strain curves of scaffolds were recorded. The Young’s modulus was calculated based on the slope of the stress-strain curve in the elastic region.

2.4 In vitro release of resveratrol



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The weighted scaffold (20 mg) was soaked into 1ml PBS buffer (resveratrol solubility 0.03 mg ml–1 in water). Samples (n=3) were then incubated at 37 °C. At each time point, supernatants were collected and stored at 4 °C for analysis, followed by adding 1 ml fresh PBS. The resveratrol in the supernatant was measured by fluorescence intensity at 330 nm using a PerkinElmer EnSpire (PerkinElmer LLC). A standard curve was carried out using a series of known resveratrol contents in 50% ethanol/PBS solution.

2.5 The effect of resveratrol on HUVEC function 2.5.1 Cell proliferation The electrospun PCL mats without or with resveratrol (4 mg) were prepared into disks (inner diameter = 10 mm), then placed into wells of 48-well tissue culture polystyrene plate (TCPS), and then fixed by hollow plexiglass molds (inner diameter = 8 mm) to prevent floating. For cell culture, the mats were sterilized by ethylene oxide. HUVECs were grown in endothelial cell medium (ECM) containing 5% fetal calf serum (FCS) and then harvested when 80% confluency was reached. After HUVECs were seeded at a density of 6×103/well in 300 µL media (n=5). The media were changed every two days. At the determined time points (1, 3 and 5 days), Cell Counting Kit-8 assay (CCK-8 assay, Beyotime) was carried out.26 In detail, the media of each well was removed and replaced with 220 µL CCK8 dilution containing 20 µL CCK8 and 200 µL ECM, and then maintained for 3 h at 37 °C. Subsequently, 100 µL supernatant was taken and added in a 96 well plate. The optical density (OD450) value was measured with the Microplate Reader (Bio-Rad). 2.5.2 NO production The NO level was measured by assaying the nitrite (a stable nitric oxide breakdown product) concentrations in culture supernatants using Griess assay.27-28 Briefly, 50 µL supernatant samples were incubated with 50 µL Griess reagent I, followed by adding 50 µL Griess reagent II (Beyotime, China). Formation of diazonium salt was monitored at 540 nm. A standard curve was conducted by a series of known concentration of NaNO2.


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2.5.3 In vitro wound-healing cell migration assay Preparation of extraction of scaffolds: 100 mg scaffold materials were weighed, and immersed into 5 mL ECM basal medium at 37 °C for 24 hours. Collect the extraction of the scaffolds and store at 4 °C for the following experiments. The cell migration assay was performed as reported.29 HUVECs were seeded in 24-well plates. Upon complete confluent, wounds were made with 1ml sterile pipette tips. Detached cells were washed with PBS buffer and the extraction from the different scaffolds was added. Immediately, images were taken under an inverted microscope (Nikon ECLIPSE Ti-U). After 24 h incubation cells migrating to the scratched area were photographed under an inverted microscope. The migration rate was calculated and reported as the percentage wound healing, which is equal to (wound length at 0 h -wound length at 24 h)/ wound length at 0 h ×100. 2.5.4 HUVEC tube formation assay for in vitro angiogenesis Tube

formation

assay

was

performed

according

previously

reported

procedures.30 In brief, 50 µL cold matrigel was added into each well in 96-well plate, and incubated at 37 °C. After gelling, HUVECs (1×104 cells per well) were seeded to matrigel-coated wells and cultured with extraction from the different scaffolds. Four hours later, tube formation was observed under an inverted microscope and images (n=3) in each well were randomly photographed. The observed total tube nodes formed by endothelial cells in each image were counted using ImageJ software.

2.6The effect of resveratrol on macrophage in cell culture RAW264.7 macrophages were grown in DMEM media containing 10% fetal bovine serum (FBS). Circular scaffolds (3 cm in diameter) were prepared and then placed in 6-well plates. Raw264.7 macrophages were seeded onto the scaffolds at a density of 4.5 × 105/well. After 24h, the supernatants were collected and the levels of TNF-α were determined by ELISA following the manufacturers’ recommendations and quantified using an ELISA reader. TRIzol reagent (Ambion) was used for RNA isolation. Then RNA concentrations were determined by SimpliNano (Biochrom). Reverse transcription



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was carried out by use of Reverse Transcription System (Promega). Quantitative PCR (Q-PCR) assay was done using LightCycler 480 SYBR green (Promega) and analyzed using Q-PCR instrument (ABI7500). RAW264.7 cells cultured on the PCL scaffold were used as control. Gene expression was analyzed using the 2-△△Ct method.24

2.7 In vivo implantation The animal experiment was allowed by the Center of Tianjin Animal Experiment Ethics Committee and Authority for animal protection (Approval No.SYXK (Jin) 2011-0008). Rat abdominal artery replacement model was used to assess the performance of the vascular grafts in situ. The procedure was carried out as described previously.25 In brief, Sprague-Dawley rats (male, weight 280-320 g) were used. Heparin (100 Units/kg) was injected through tail veins for anticoagulation before surgery. The tubular vascular grafts (2.0 mm in inner diameter, 500 µm in thickness and 1.0 cm in length) were used as the artery substitutes. No anticoagulation drug was administered after surgery. The patency of the grafts was checked two weeks and four weeks after implantation. After sacrifice, grafts were explanted and transected into two parts from the middle. One part was for frozen cross-section and the other was longitudinally cut into two pieces. One piece was for stereomicroscope observation and SEM examination. The other piece was embedded in OCT with liquid nitrogen for frozen longitude-section for CD31 staining.

2.8. Analysis of the explanted vascular grafts 2.8.1. Scanning electron microscopy The longitudinal pieces of grafts were first observed under stereomicroscope and then fixed with 2.5% glutaraldehyde overnight and then rinsed using water, subsequently dehydrated by graded ethanol. Samples were air-dried and placed onto aluminum stubs. After sputter-coated with gold, they were finally observed under SEM (Quanta200, Czech). 2.8.2. Histological staining


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Samples were fixed with 4% paraformaldehyde for 4h, and then dehydrated by 30% sucrose solution. For frozen sections, samples embedded in OCT at -20 °C were cut into 6-µm slices. Haematoxylin & eosin (H&E) staining was performed to examine the neo-tissue formation of the explanted, and images were photographed by an upright microscope (Olympus BX53). 2.8.3. Immunofluorescence staining Endothelial cell was identified using anti-CD31 (1:70, Abcam) antibody and smooth muscle cell was identified using mouse anti-a-SMA (a-SMA, 1:100, Abcam) and mouse anti-smooth muscle myosin heavy chain I (SM-MHC, 1:100, Abcam) antibodies. In addition, anti-mannose receptor antibody (CD206, 1:200, Abcam) was used to identify M2 macrophages. Alexa Fluor 488 goat anti-mouse IgG (1:200, Invitrogen) and goat anti-rabbit IgG (1:200, Invitrogen) were used as the secondary antibodies, respectively. The protocol for immunofluorescence staining referred to our previous report.25 In brief, sections were first fixed by cold acetone for 5 min. Following washed by PBS, the sections were blocked by 5% normal goat serum for 30 min at room temperature, followed by incubation with primary antibodies in PBS overnight at 4 °C. On the second day, the sections were rinsed by PBS for 5 times, and then incubated with secondary antibody in PBS for 2 h at room temperature. After washing with PBS, samples were counterstained with 4, 6-diamidino-2-phenylindole (DAPI, DapiFluoromount G, Southern Biotech). Images were recorded by a fluorescence microscope (Leica DM3000). Endothelial coverage was quantified by the length of endothelial cell layer on the luminal surface and the percentage of it to the total graft length. The average thickness of SMCs layer calculated and reported as the area of SMCs layer divided by the length of SMCs layer. The density of capillaries was measured as capillaries per high power field (HPF, ×200).

2.9 Statistical analysis Data were given as the mean ± standard error of the mean. The two-tailed unpaired t-test was performed to compare differences between two groups. One-way ANOVA was used to compare differences among three or more groups. The statistical



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significance was defined as p < 0.05.

3. Results 3.1 Morphology and mechanical property of scaffolds Scaffolds were fabricated by electrospinning PCL solution containing resveratrol. The fibers of the scaffolds were smooth and aligned randomly in SEM images (Figure1A-D). The average diameter of the electrospun fibers showed a slight decrease as the resveratrol concentration increased (Table 1). In detail, the fiber diameter was 5.1±0.9 µm in PCL-0.5%res group and 6.0±1.3 µm in PCL-1.0%res group, which is smaller than that in PCL-0.25%res group (6.7±0.7 µm). In accordance with the diameter changes among scaffolds, the pore size showed the same trend as 32.9±7.2 µm in PCL-0.25%res, 26.2±6.5 µm in PCL-0.5%res and 27.4±6.2 µm in PCL-1.0%res. The stress-strain curves of the scaffolds were shown in Figure 1E.The elastic modulus was around 10 Mpa in the PCL scaffolds (Figure 1F), and it was 8.3±0.2 Mpa, 7.0±0.2Mpa, 5.0±0.3 Mpa for PCL-0.25%res, PCL-0.5%res and PCL-1.0%res group, respectively, showing a decreasing trend with the increase of resveratrol content in the scaffolds (Figure 1F). The ultimate tensile strength had the identical trend as the elastic modulus among different groups. In details, PCL-0.25% res (1.6±0.1 Mpa) had the highest stress, then PCL-0.5%res (1.3±0.1 Mpa) following by PCL-1.0%res (1.0±0.0 Mpa). No significant difference existed in the strain with values around 600%, as seen in Figure 1H. In all, compared with that of native arteries,22 the mechanical properties of these scaffolds were strong enough to endure the blood pressure and stress from the adjacent blood vessels.

3.2 The release of resveratrol The representative release curve of resveratrol from scaffolds was shown in Figure 2. The release of resveratrol was in a sustained manner. The release rate was fastest in the initial 24 hours, then slowed down to a steady rate in the following days. The amount of cumulative release (Figure 2A) was 6.7±1.0 µg, 22.2±1.4 µg, 30.9±4.2


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µg after day 1, and 11.3±2.0 µg, 40.2±2.9 µg, 58.3±3.5 µg at day 7 in PCL-0.25% res, PCL-0.5% res and PCL-1.0% res, respectively. At day 7, the percentage of cumulative release (Figure 2B) was about 40% in both PCL-0.25% res and PCL-0.5% res group, while it was slightly less in PCL-1.0% res group. No burst release was observed in all groups during the detection period.

3.3 The effect of resveratrol on HUVECs function No significant difference was shown among the four groups regarding the viability of HUVECs (Figure 3A). The effect of resveratrol on NO production of HUVECs was tested by Griess reagent assay (Figure 3B). NO production in the PCL group was 3.7±0.7 µM on the first day, increased to 4.9±0.5 µM on the third day, which was similar to that in the groups with resveratrol. However, NO production increased in all scaffolds with resveratrol at day 5. The value was 7.1±0.3 µM in PCL-0.5% res and 6.9±0.5 µM in PCL-1.0% res, which was apparently higher in the scaffolds with resveratrol, compared to that in the PCL (5.1±0.2 µM). Our results indicated that resveratrol could promote HUVECs to produce more NO. Moreover, we examined whether the scaffolds with resveratrol could improve endothelialization and tubulogenesis in vitro by wound scratch and tube formation assays. In wound scratch assay (Figure 4D), cells migrated 26.3±6.9% of the initial wound distance in the PCL group, while distance of cell migration increased to 36.3±7.1% in PCL-0.25% res, and 46.1±7.4% in PCL-0.5% res. However, it decreased to 34.8±9.4% when the concentration of resveratrol was increased to 1.0% (Figure 4I). These results suggested that the effect of resveratrol was dose-dependent. In tube formation assay as seen in Figure 4 E-H, more nodes were found in all the scaffolds with resveratrol compared with the PCL group (28±2 per field). In addition, we also found that nodes in PCL-0.5% res group (59±3) was more than the other two resveratrol groups, 51±1 in PCL-0.25% res and 46±4 in PCL-1.0% res (Figure 4J). These data demonstrated that scaffolds with resveratrol could dramatically improve HUVECs function such as endothelial cell migration and tube formation, and this effect was on dose-dependent.



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3.4 The effect of resveratrol on macrophage polarization and pro-inflammation Real-time PCR assay was used to assess the effect of resveratrol on mRNA expressions of macrophages with different phenotypes. The mRNA expression of IL-1b and iNOs, related to the M1 phenotype, were evidently decreased, whereas genes associated with the M2 phenotype, including ArgI and FiZZ1, were highly expressed in cells cultured on the resveratrol-loaded mat compared to those on the PCL mat at day 1 (Figure 5A). These findings suggest that resveratrol-loaded scaffolds could stimulate macrophage polarization toward the M2 phenotype. Further, TNF-α expression was examined as the main pro-inflammatory factor to reflect the effect of resveratrol on macrophage. The concentration of TNF-α was 60 pg/mL in the PCL group, whereas it was undetectable in all the scaffolds with resveratrol

(Figure

5B).

It

verified

that

resveratrol

could

down-regulate

pro-inflammation reaction by reducing TNF-αsecretion.

3.5 The performance of the PCL-0.5%res graft as a rat abdominal artery substitute Based on the data from in vitro, the PCL-0.5%res group was optimal in regulating endothelial cell function. As a result, we chose the PCL-0.5% res tubular scaffold for in vivo performance assessment as a rat abdominal artery substitute (Figure 6). The PCL scaffold was used as the control. The implants were harvested at 2 weeks and 4 weeks. Every group had five parallel rats at each time point. All scaffolds were patent in both groups at all examined time points without an aneurysm and bleeding. The inner surface of the scaffolds observed by stereomicroscope was smooth and free of thrombi. Meanwhile, the scaffolds integrated well with the adjacent blood vessels as shown in Figure 6E and F. H&E staining showed that the scaffolds were patent with little neo-tissue formation (Figure 6A-D). No significant difference was present in the inner diameter between the two groups at 2 weeks and 4 weeks (Figure 6G). Cell infiltration was quantitatively analyzed based on DAPI stained cross sections (Figure 7A and B). There is such a trend that the density of cells increased from the luminal side to the adventitia side. However, no difference was


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observed between the two groups in each region (Figure 7C), which suggests resveratrol has no effect on cell infiltration (Figure 7D). The endothelium plays a key role in maintaining vascular homeostasis, such as anti-thrombosis and prevention of restenosis caused by smooth muscle cells malignant proliferation. Rapid endothelialization is crucial to guarantee good performance of artificial small diameter vascular scaffolds after in situ implantation. Herein, endothelial

formation

was

demonstrated

by

SEM

observation

and

immunofluorescence staining by CD31. A neo-tissue layer was found on the surface of the scaffolds (Figure 8A) with cobblestone like cells aligning along with the blood flow direction. CD31 staining confirmed that the monolayer consists of endothelial coverage was 80%, while it was only 60% in the PCL group (Figure 8C). Such a significant difference indicates that resveratrol could promote endothelialization. In addition, we examined the effect of resveratrol on smooth muscle cells regeneration by immunofluorescence staining with α-SMA and SM-MHC antibodies. As shown in Figure 9, the average thickness of both α-SMA and SM-MHC positive cells was almost the same in both groups. It suggests that resveratrol has no effect on the regeneration of vascular smooth muscle cells.

3.6 The effect of resveratrol on capillary formation We checked capillary formation within the scaffolds wall by CD31 antibody staining. As shown in Figure 10, the density of capillaries in the PCL-0.5%res group (Figure 10B and D) was distinctly more than that in the PCL group (Figure 10A and C) at both 2weeks and 4 weeks.

3.7 The effect of resveratrol on macrophage polarization The M2 macrophages were analyzed by CD206 antibody staining. As shown in Figure 11, the density of the M2 macrophages in the PCL-0.5%res scaffolds is greater than that in the PCL scaffolds at 2 weeks. However, it decreased to the same level as the control group at 4 weeks. It suggests that resveratrol induced early macrophage polarization, which may lead to enhanced repair and neo-tissue regeneration.



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4. Discussion Although intensive researches have been devoted to developing small diameter vascular grafts as an artery substitute for cardiovascular disease treatment, the challenge remains. The lack of rapid endothelialization often results in acute thrombosis and intimal hyperplasia. In the present study, we aimed to develop a bioactive electrospun small diameter vascular graft by loading with resveratrol. The scaffold could release resveratrol in a time-dependent manner, regulate HUVECs functions and macrophage behaviors. Its feasibility as small diameter vascular graft was demonstrated in a rat abdominal artery replacement model, in which the resveratrol-loaded grafts showed rapid endothelialization, enhanced capillary formation and reduced inflammatory. Resveratrol released from the PCL electrospun scaffolds in a sustained manner over a long term (Figure 2). Resveratrol is a small molecular drug with high hydrophobicity. The release kinetics of resveratrol-loaded PCL electrospun scaffolds were in a similar way with those of paclitaxel-loaded polycaprolactone films31 and the DPA-loaded BPU scaffolds.32 The fast initial release at first hours could result from the low glass transition temperature of PCL, while the low release over 1 week may be attributed to the hydrophobic interaction between the resveratrol and PCL. The endothelium plays a critical role in maintaining vascular homeostasis and prevention of vascular diseases. Rapid endothelialization is essential for the development of vascular grafts. Various approaches have been developed. For example, tissue engineered vascular grafts with pre-implanted cells including endothelial progenitor cells,1 mesenchymal stem cells (MSCs)

33

and iPS-ECs.34 In

addition, cell-free vascular scaffolds modified with bioactive molecules2 have been prepared to promote rapid vascular endothelialization of artificial blood vessel. Besides, scaffolds with capable of gene delivery were prepared to promote endothelial cell regeneration through local transfection of cells around the scaffolds to release angiogenic factors.4 In this study, we utilized resveratrol to modify vascular scaffolds, and it demonstrated accelerated endothelialization in a rat abdominal artery implantation. The advantages of resveratrol are inexpensive, easily produced and long


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shelf life compare to cytokines and pre-implanted cells, which are beneficial for commercial production. Importantly, resveratrol showed multi-targets and high activity in tissue repair and wound healing. The positive effect of resveratrol on endothelialization could be explained by the role of resveratrol on ECs’ migration and tube formation. However, the impact of resveratrol on EC activity was dose-dependent. Thus, it exerts positive impact on EC migration and tube formation at low concentration whereas when administrated in high dosage, it inhibits EC activities. These results are consistent with the previous report in which resveratrol promotes angiogenesis at a low dose, while anti-angiogenesis effect in cancer at high concentration.35 Interestingly, we also found that the dose-dependency of resveratrol was not consistent between HUVECs and macrophages. It might be due to the different nature of the two cell types. In addition, we further confirm that resveratrol promotes NO production, as reported previously.36-37 The mechanism that underlines the effect of resveratrol on endothelial NO is still unclear. Several signaling pathways might be involved, such as NAD+,38 SIRT1,39-40 AMPK,41 Nrf2,42 and ER.43 In addition, resveratrol was reported to play a role in inhibiting inflammatory reaction44-47 and macrophage polarization.48 As reported, resveratrol could reduce LPS induced pro-inflammatory responses.49 In accordance, we found that the M1 macrophage related genes (iNOs and IL-1b) was down-regulated while the M2 macrophage related genes (FiZZ1 and Arg I) were up-regulated in the resveratrol-loaded scaffolds compared to the control group. For cytokine release experiments, we found that resveratrol dramatically decreased the TNF-α secretion.50-51 In addition, more CD206+ cells, the anti-inflammatory macrophages, were detected within the scaffolds with resveratrol (Figure 11). These data indicated that the enhanced vascular regeneration could be at least be partially explained by the beneficial resveratrol in balancing the inflammation reaction. Additionally, resveratrol was reported to exert positive effect in inhibiting intimal hyperplasia.52-54 However, we did not observe this effect in the present study (Figure 9). It may be due to the difference in the regeneration/repair processes between the vascular grafts and the vascular injury models. First, the initial amount of smooth



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muscle cells in the two models was different. No smooth muscle cells exist in the PCL vascular grafts used in our study. Second, SMCs regeneration is considered advantageous for vascular graft implantation, since they play a critical role in vascular contraction and relaxation, extracellular matrix composition as well as maintaining endothelial cell stability.55 However, SMC proliferation in the vascular injury model is adverse because it causes intimal hyperplasia and restenosis. Third, 4 weeks may not be long enough to study the effect of resveratrol on inhibiting SMC proliferation. The capillaries play an important role in the regeneration process, especially at the early stage. Scaffolds loaded with resveratrol demonstrated enhanced capillaries formation at 2 weeks (Figure 8), and there is a slight decline of the capillary vessel formation in the PCL/Res group at 4 weeks. Although the number of small capillaries was not as abundant as those observed at 2 weeks, these vessels seem much more mature with large diameters. Besides, the number of M2 macrophages decreased at 4 weeks, which may be due to the decline of resveratrol release from 2 weeks to 4 weeks. Although such a decrease occurred in the PCL/Res group, the number and maturity of capillaries in all time points are also better than the PCL group, which make it possible for wider applications, not only limited to small diameter vascular grafts but also in other ischemia situations, such as myocardium patch, low limb ischemia, islets transplantation and nerve repair. The scaffolds are also promising as stem cell delivery platform in stem cell therapy field.

5. Conclusion In summary, we have developed a vascular graft that could release resveratrol in a sustained and controlled manner. The scaffold could improve endothelial cell functions including tube formation, migration and NO production. Meanwhile, the scaffold could diminish the inflammatory reaction by reducing TNF-α secretion, up-regulating the expression of M2 related genes and increasing the M2 macrophages within the scaffold. Furthermore, the scaffolds with resveratrol exhibited excellent performances as small diameter vascular grafts in rat abdominal artery replacement model, including rapid endothelialization and vascularization. In summary, resveratrol


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can be used as a potential bioactive molecular for modification of vascular grafts or other scaffolds that could have broad application potential in tissue engineering and regenerative medicine.

Acknowledgments The work was financially supported by NSFC projects (81530059, 31400833, 81601625, 81401534), Science & Technology Projects of Tianjin of China (no. 16JCQNJC14100 and 14JCQNJC13900), Program for Changjiang Scholars and Innovative Research Team in University (IRT13023), Program for Innovative Research Team in Peking Union Medical College, China Postdoctoral Science Foundation (No.2016M590197).

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Table 1. Scaffold physical properties

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Figure 1, morphology and mechanical properties of scaffolds. SEM images showed that all the scaffolds consist of random fibers with uniform diameter.The fiber diameter showed a trend of decrease as the resveratrol-loaded concentration increased. (A) PCL, (B) PCL-0.25%res, (C) PCL-0.5%res, (D) PCL-1.0%res. E, the representative stress-strain curves of the scaffolds. Quantitative data for elastic modulus, maximum strain and maximum stress was calculated and shown in as F, G and H. 94x88mm (300 x 300 DPI)



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Figure 2, resveratrol release curves. A, the cumulative release amount of resveratrol in different scaffolds. Resveratrol was released in a sustained manner. B, the percentage of cumulative release 78x99mm (300 x 300 DPI)


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Figure 3, the effect of resveratrol on HUVECs proliferation and NO production. A, No significant difference was observed between the four groups regarding HUVECs proliferation. B, Scaffolds with resveratrol could promote the production of NO by endothelial cells. 234x99mm (300 x 300 DPI)



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Figure 4, the effect of resveratrol on HUVECs in angiogenesis. Crystal violet staining showed the migration of HUVECs on scaffolds (A-D), scale bar：200µm. Bight field microscope images showed the tube formation in different scaffolds (E-H), scale bar：500µm. PCL (A, E), PCL-0.25%res (B, F), PCL-0.5%res (C, G), PCL1.0%res (D, H). I, the distance of cell migration in wound scratch assay. J, the number of nodes per field in tube formation. Scaffolds with resveratrol could promote HUVECs migration and tube formation, compared with the PCL scaffold. In addition, the effect was dose-dependent and PCL-0.5%res group showed the optimal effect. 127x99mm (300 x 300 DPI)


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Figure 5, the effect of resveratrol on macrophage polarization (A). The M1 macrophage phenotype related genes were down-regulated, whereas M2 macrophage related genes were up-regulated on scaffolds with resveratrol. (B), TNF-α secretion from macrophages was diminished on scaffolds with resveratrol comparison with that on the PCL group. 239x95mm (300 x 300 DPI)



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Figure 6, the gross morphology and H&E staining of explanted grafts at 2 weeks (A, C) and 4 weeks (B, D). The inner surface of the scaffolds observed by stereomicroscope (E, PCL and F, PCL-0.5%res) was clean and free of thrombi. No significant difference in the luminal diameter was observed between two groups (G). 155x99mm (300 x 300 DPI)


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Figure 7, the effect of resveratrol on cell infiltration. Immunofluorescence images of DAPI staining in both groups (A and B). C, the schematic diagram of the cell density for analysis. D, Cell density was almost the same in the PCL-0.5%res group and PCL group. There is a trend that the density of cells increased from the luminal side to the adventitia side. 130x99mm (300 x 300 DPI)



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Figure 8, Resveratrol promoted endothelialization. A, SEM images of the luminal surface of scaffolds at 4 weeks. B, representative images of immunofluorescent staining by CD31 antibody. The digit labeled images were the magnification of red boxes in the images. At four weeks after implantation, the luminal surface was covered about 80% by CD31+ cells in the resveratrol group, whereas it was about 60% in the PCL scaffolds(C). The length of CD31+ cells in the resveratrol group was also longer than the PCL group (D). 86x99mm (300 x 300 DPI)


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Figure 9, Regeneration of smooth muscle cells. Immunofluorescence images of α-SMA (A, B) and SM-MHC (C, D) staining. There is no significant difference in the average thickness of α-SMA (E) and SM-MHC (F) between two groups. 182x99mm (300 x 300 DPI)



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Figure 10, Capillary formation. Representative fluorescence images of CD31 stained capillaries in the vascular graft wall at 2 weeks (A, B) and 4 weeks (C, D). (E) Quantification of capillary density. The vessel density significantly increased in the PCL-0.5%res group（A and C） compared with that in the PCL group（B and D） at 2 weeks and 4 weeks. 76x99mm (300 x 300 DPI)


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Figure 11, the effect of resveratrol on macrophage polarization. Immunofluorescence images of CD206 antibody staining of PCL (A, C) and PCL-0.5%res (B, D) at two time points. The number of CD206+cells was shown in E. Resveratrol could promote M2 macrophage fast infiltration into the scaffold walls at 2 weeks. This effect was not observed at 4 weeks. 73x99mm (300 x 300 DPI)



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Graphical Abstract 156x47mm (300 x 300 DPI)


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Effect of Resveratrol on Modulation of Endothelial Cells and

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