The placental growth factor attenuates intimal hyperplasia in vein grafts by improving endothelial dysfunction

Journal Pre-proof The placental growth factor attenuates intimal hyperplasia in vein grafts by improving endothelial dysfunction Jian Zhang, Jun Shi, Hao Ma, Lulu Liu, Li He, Chaoyi Qin, Dengshen Zhang, Yingqiang Guo, Renrong Gong PII:

S0014-2999(19)30808-8

DOI:

https://doi.org/10.1016/j.ejphar.2019.172856

Reference:

EJP 172856

To appear in:

European Journal of Pharmacology

Received Date: 5 March 2019 Revised Date:

6 December 2019

Accepted Date: 9 December 2019

Please cite this article as: Zhang, J., Shi, J., Ma, H., Liu, L., He, L., Qin, C., Zhang, D., Guo, Y., Gong, R., The placental growth factor attenuates intimal hyperplasia in vein grafts by improving endothelial dysfunction, European Journal of Pharmacology (2020), doi: https://doi.org/10.1016/ j.ejphar.2019.172856. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

The placental growth factor attenuates intimal hyperplasia in vein grafts by improving endothelial dysfunction

Jian Zhang1,2,†, Jun Shi1,†, Hao Ma1, Lulu Liu1, Li He1, Chaoyi Qin1, Dengshen Zhang1, Yingqiang Guo1,*, Renrong Gong3,* 1

Department of Cardiac Macrovascular Surgery, West China Hospital, Sichuan

University, Chengdu, Sichuan, P. R. China 2

Department of Cardiovascular Surgery, Affiliated Hospital of Zunyi Medical

University, Zunyi, Guizhou, China 3

Anesthesia Surgery Center, West China Hospital, Sichuan University, Chengdu,

Sichuan, P. R. China †

These authors contributed equally to this work.

*

Corresponding Author:

Yingqiang Guo Department of Cardiac Macrovascular Surgery, West China Hospital, Sichuan University, Chengdu, Sichuan, P. R. China Renrong Gong Anesthesia Surgery Center, West China Hospital, Sichuan University, Chengdu, Sichuan, P. R. China Tel: +86-18980601876 Fax: +86-28-85421833 E-mail: [email protected]; [email protected]

1

ABSTRACT Saphenous vein grafts (SVG) patency is limited by intimal hyperplasia (IH) caused by endothelial dysfunction. This study aimed to explore the effect of placental growth factor (PlGF) on the endothelial function of SVG. In rat models of external jugular vein-carotid artery graft treated with PlGF or saline hydrogel, PlGF inhibited vein graft IH (day 28: 12.0±1.9 vs. 61.7±13.1 µm, P<0.001), promoted microvessel proliferation (day 14: 33.3% 3+ vs. 50.0% 2+, P=0.03), and increased nitric oxide (NO) production (P<0.05 on days 1/3/5) and NO synthase (NOS) expression by immunohistochemistry. In human umbilical vein endothelial cells (HUVECs) cultured under hypoxia and treated or not with PlGF, PlGF restored the survival (50 ng/ml PlGF, 48 h: 91.7±0.6% vs. 84.9±0.5%, P<0.01), migration (by Matrigel assay), and tube formation ability (junctions, tubules, and tubule total length; all P<0.01) of HUVECs after hypoxia. PlGF increased NO production through increased eNOS expression (P<0.05), without changes in iNOS expression. The mRNA expression of eNOS decreased after the addition of the PI3K inhibitor LY294002 (P<0.05). PlGF promoted the protein expression of eNOS by up-regulating AKT, and the AKT and eNOS protein levels were decreased after adding LY294002 (all P<0.05). In conclusion, PlGF is a candidate for the inhibition of IH in SVG after coronary artery bypass graft. The effects of PlGF are mediated by the upregulation of the eNOS mRNA and protein through the PI3K/AKT signaling pathway. PlGF promotes the secretion of NO by endothelial cells and thereby reduces the occurrence and development of IH.

Key words: saphenous vein grafts; coronary artery bypass graft; placental growth factor; intimal hyperplasia; nitric oxide; PI3K.

2

1. INTRODUCTION Autologous saphenous vein grafts (SVG) remains widely used in coronary artery bypass surgery (CABG) (Gaudino et al., 2018), particularly in multi-vessel disease (Lopes et al., 2012), but no suitable arterial graft can be prepared for all vessels (Kahraman et al., 2016). Disappointingly, the effectiveness of SVGs remains limited due to poor long-term patency rates (Goldstone et al., 2016; Zhao et al., 2018). The poor long-term outcomes of SVG are due to the luminal narrowing that results from intimal hyperplasia (IH), medial thickening, and subsequent superimposed accelerated atherosclerosis (Davies and Hagen, 2011; Goldman et al., 2011; Sabik et al., 2005). IH is a complex process that involves early phase endothelial injury and inflammation due to the harvesting and implantation of the SVG (de Vries and Quax, 2018; Lardenoye et al., 2002) and to the exposure of the venous endothelium to high blood pressure and wall shear stress (Meirson et al., 2015; Tseng et al., 2014). IH is necessary to the process of arterialization, i.e., vein graft adaptation to the arterial conditions (Muto et al., 2010), but IH will ultimately lead to atherosclerosis that will compromise graft patency (Motwani and Topol, 1998). The exact molecular mechanisms are complex, and some factors are still poorly known. The current preventive strategies for the prevention of SVG IH include aspirin, clopidogrel, anticoagulants, ticagrelor/prasugrel, lipid-lowering therapy, and gene therapy (McKavanagh et al., 2017). The primary cause of SVG stenosis is endothelial dysfunction (Li et al., 2013; Valdes and Diaz, 2018). Compromised bioavailability of nitric oxide (NO) is central in endothelial dysfunction and is due to increased oxidative stress, decreased NO synthase (NOS) expression and activity, and secretion of various cytokines (Prieto et al., 2014). NO is a potent vasodilator and an inhibitor of platelet aggregation

3

(Tousoulis et al., 2012). The PI3K/Akt signaling pathway is involved in the regulation of NOS activity and NO production, as well as in angiogenesis (Fulton et al., 1999; Kawasaki et al., 2003). The placental growth factor (PlGF) binds to the vascular endothelial growth factor (VEGF) receptor 1 (VEGFR1) and enhances the biological efficiency of VEGF, including stimulating endothelial cell migration and survival, and promoting angiogenesis and arteriogenesis (Taimeh et al., 2013). PlGF is a critical factor regulating vasculogenesis under physiological and pathological conditions without affecting the healthy blood vessels (Dewerchin and Carmeliet, 2014; Luttun et al., 2002). PlGF is rapidly produced in infarct myocardium and improves left ventricular function in the patients (Iwama et al., 2006). PlGF does not increase vascular permeability (Gaal et al., 2013; Zheng et al., 2007), and PlGF induces less inflammation than VEGF (Gaal et al., 2013). Whether PlGF can be used as a therapeutic approach against IH and CVG patency failure remains to be determined. Therefore, we hypothesized that PlGF, as a potent angiogenic factor, can effectively inhibit IH in SVG after CABG. The aim of the present study was to explore the effect of PlGF on the endothelial function of SVG. This could provide an innovative method for the management of IH after CABG and to improve the patency of SVG.

2. MATERIALS AND METHODS 2.1. Vein grafting models and study design Adult male Sprague-Dawley (SD) rats (specific pathogen-free grade) weighing 200-250 g were used in this study. All protocols were approved by the animal care and use committee of the West China Hospital of Sichuan University. The animal procedures conformed to the Guide for the Care and Use of Laboratory Animals (NIH

4

Publication No.85-23, revised 1996). Supplementary Fig. S1 presents the surgery process. The rats were anesthetized with an intraperitoneal injection of 3% pentobarbital sodium (0.5 ml/100 g). Operations were performed with a surgical microscope (World Precision Instruments, Sarasota, FL, USA) under sterile conditions. A 3.0-cm segment of the right external jugular vein was carefully dissected and excised through a midline neck incision, washed with saline to remove blood clots, and immersed in heparinized saline (1 ml of 0.9% saline solution with heparin 5 IU). After exposure and clamping of the right common carotid artery (RCCA), the RCCA was dissected, and the vein was placed as an interposition graft in a reversed manner. The adventitia of the vein grafts was injected with 0.1 ml of liquid hydrogel containing either 50 ng PlGF (PeproTech, Rocky Hill, NJ, USA; catalog #100-06, lot #0708307) or saline. For Pluronic® F-127 hydrogel preparation, 30 g of Pluronic® F-127 was weighed and added to 100 ml of sterile water. The mixture underwent magnetic stirring in an ice bath until the powder was completely dissolved. After sterilization by filtration with a 0.22-µm filter on a clean bench, the gel was separated into 9-ml aliquots in 20-ml centrifuge tubes and kept at 4°C until use. The gel is in a liquid state at 0-4°C and solidifies at >15°C. The wound was closed in layers. The rats were randomized into three groups: (i) normal group (skin incision without vein grafting), n=6; (ii) control group (hydrogel containing saline), n=24; and (iii) PlGF group (hydrogel containing PlGF), n=24.

2.2. Successful establishment of the animal model The average operation time was 60 min. The mean venous ischemia time was 36 min. Finally, 54 rat models were successful. One rat in the control group suddenly died on the first day after the operation, and autopsy found massive blood accumulation in the

5

abdominal cavity, which may have been caused by the anesthesia or the surgery. In the PlGF group, a thrombus was observed in one rat on the third day. In the control group, a thrombus was observed in one rat on the 14th day. No obvious behavioral abnormalities were found in the rats. Bleeding from the transplanted blood vessels occurred in one rat on the 3rd day in the PlGF group.

2.3. Sample collection Vein grafts were collected at 3, 7, 14, and 28 days post-operation from the control and PlGF groups (n=6/group/day) for enzyme-linked immunosorbent assay (ELISA), histopathology with hematoxylin & eosin (H&E) staining, immunohistochemistry and NO nitrate reductase assay (n=6). Vein tissues collected from the normal group on the day of sham operation were used as the normal control in the ELISA. For obtaining the vein grafts/tissues, the chest was opened after anesthesia to expose the heart, 0.2 ml of 1% heparin sodium was injected into the left ventricle, and aortic cannulation was performed through the left ventricle. After fixation by vessel forceps, the right auricle was cut; 150 ml of normal saline were used for fast washing of the vessels, and then 250 ml of 0.01 mol LPBS (4°C, pH 7.40) with 4% paraformaldehyde were used for perfusion fixation, which was fast first and then slow, for a total time of 50 min. After the samples were collected, the rats were killed using 10% chloral hydrate. The middle segment of the vein graft (or vein tissue) for each rat was used to prepare a section (2015 type rotary slicer, Leica Instruments Co., Ltd., Wetzlar, Germany).

2.4. Immunohistochemistry The sections were conventionally dewaxed. Endogenous peroxidase was inactivated

6

at room temperature for 10 min using hydrogen peroxide and distilled water at 1:10 and washed with distilled water thrice. The sections were immersed in 0.01 M citrate buffer (pH 6.0) and heated in a microwave oven for antigen retrieval. It was repeated a second time after 5 min. After cooling, PBS (pH 7.2-7.4) was used to wash the sections twice. Goat serum blocking buffer (batch number: 13152A11, Beijing Zsbio Co., Ltd.) was added and incubated at room temperature for 20 min. Rabbit anti-eNOS polyclonal primary antibody (1:200; ab76198, UK Abcam (Shanghai) Trading Co., Ltd.) or rabbit anti-iNOS polyclonal primary antibody (1:200; ab15323, UK Abcam (Shanghai) Trading Co., Ltd.) was added. Anti-CD31 (1:200; Abcam) and anti-PIGF (1:100; Abcam) primary antibodies were also used. They were incubated overnight at 4°C and washed three times with PBS. Biotinylated goat anti-rat/rabbit IgG secondary antibody (13152A11, Beijing Zsbio Co., Ltd.) was added and incubated at 37°C for 30 min and washed thrice with PBS. Horseradish peroxidase-labeled streptavidin (HRP/A-V; 13152A11, Beijing Zsbio Co., Ltd.) was added, incubated at 37°C for 30 min, and washed four times with PBS. The DAB color development kit (K135925C, Beijing Zsbio Co., Ltd.) was used for color development at room temperature. Hematoxylin was used for light counterstaining. The sections were dehydrated, cleared, and mounted with neutral gum.

2.5. Image acquisition and analysis After H&E or immunohistochemistry, one field of view was captured for each section. The results of rats (n=6) in each group at each time point were analyzed. The intimal and medial areas were calculated using the Image-Pro Plus 6.0 image analysis system (Media Cybernetics, Inc., Rockville, MD, USA). Images were acquired using the BA200 Digital Trinocular Micro Camera System (MOTIC Group Co., Ltd.). First, all

7

tissues for each section were observed at 100×. Then, areas at 400× were selected and acquired according to the size and expression of the tissues. The average optical density of all images was measured using Image-Pro Plus 6.0.

2.6. Endothelial cell injury models Human umbilical vein endothelial cells (HUVECs) were selected for hypoxia culture to obtain endothelial cell injury models. HUVECs were obtained from the Core Facility of West China Hospital of Sichuan University (Sichuan, China). The cells at passage 3-4 were used. The cells were grown in low-glucose Dulbecco’s modified Eagle’s medium (DMEM; GIBCO, Invitrogen Inc., Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; GIBCO, Invitrogen Inc., Carlsbad, CA, USA), 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C. For the normoxia experiments, the HUVECs were cultured at 37°C in humidified 5% CO2/95% air. For hypoxia experiments, the HUVECs were cultured at 37°C with 5% CO2, 94% N2, and 1% O2 in a multigas incubator for 3, 6, 12, 18, or 24 h. The same hypoxic conditions were applied to all the assays with the same time length. To evaluate the hypoxic effects in HUVECs, acridine orange (AO)/propidium iodide (PI) double staining was used to monitor survival rate. The Cell Counting Kit-8 kit (CCK-8) assay was used for cell viability.

2.7. Acridine orange/propidium iodide staining and flow cytometry To identify the effects of PlGF on cell survival in endothelial cell injury models, HUVECs were seeded in three 6-well plates (25,000 cells/well) and cultured in DMEM medium containing 10% FBS overnight. The cells were synchronized for 24 h by starvation in DMEM medium containing 1% FBS. After synchronization, the

8

culture medium was replaced by normal medium (containing 10% FBS), and two plates were put into a multigas incubator for 18 h of hypoxic culture. The remaining plate was cultured under normoxia. After hypoxic culture, PlGF was added at final concentrations of 0, 10, 50, or 100 ng/ml, and all cells were cultured under normoxia for 48 h. The cells were collected and stained with an AO/PI kit (BestBio Inc., Shanghai, China) using standard procedure (Bank, 1988). The results were obtained by fluorescence microscopy and flow cytometry. All assays were performed in triplicates.

2.8. Cell counting kit-8 assay CCK-8 Dojindo Laboratories, Kumamoto, Japan) was used to detect the effect of PlGF on cell proliferation. Briefly, the cells in the hypoxia group after culture with different concentrations of PlGF (0, 10, 50, or 100 ng/ml) were seeded in 96-well plates (3000 cells/well) and incubated for 24, 48, 72, 96, and 120 h. At the indicated time points, 10 µl of CCK-8 solution were added, and the plate was incubated for 2 h. A microplate reader (Bio-Tek, Winooski, VT, USA) was used to measure the absorbance at 450 nm. All assays were done in triplicates.

2.9. Transwell migration assay The migration assay was performed with a transwell system (Corning Inc., Corning, NY, USA) containing a polycarbonate membrane filter (8-µm pore size). HUVECs from different interventions (20,000 cells/well) were placed in the upper chamber, and 100 µl of complete medium were placed in the lower well to induce cell migration. After 24 h of incubation, the cells on the upper side of the membrane were removed using cotton swabs. The cells on the bottom surface of the membrane were stained

9

with 0.1% crystal violet and counted in five random fields (200×).

2.10. HUVEC tube formation assay The effect of PlGF on the formation of capillary-like structures (tubes) in endothelial cells was measured by HUVEC catheterization. Matrigel (18.3 mg/mL; BD Biosciences, Franklin Lake, NJ, USA) was diluted 1.7 times with serum-free medium and plated on a 96-well plate (50 µl/well) for 0.5 hour at 37°C. HUVECs from different interventions were added to the Matrigel-coated wells (50 µl, 20,000 cells/well) for culture for 8 h. The tube formations of HUVECs were observed using a phase-contrast inverted microscope. The Image J software (National Institutes of Health, Bethesda, MD, USA) was used to calculate the total number of junction of tube formation, the total number of tube formation, and the total length of the branches of tube formation.

2.11. ELISA and NO nitrate reductase assay The PlGF concentration in vein grafts was measured using the Rat PlGF ELISA kit (Abcam), according to the manufacturer's protocol. The tissue samples were treated in the same way as the HUVECs. NO accumulation was expressed as nitrites and nitrates, which were detected using a NO nitrate reductase assay kit (Nanjing Jiancheng Bioengineering Inc., Nanjing, China), according to the manufacturer's protocol. NOS-3 in the supernatant was detected using an ELISA kit (Cloud-Clone Corp, Wuhan, China), according to the manufacturer's protocol.

2.12. Quantitative RT-PCR Total RNA was extracted using Trizol (GeneTex Inc., Irvine, CA, USA). For each

10

sample, 1 µg of RNA was converted to complementary DNA (cDNA) using the iScript™ gDNA Clear cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). All experiments were performed according to the manufacturer’s protocol. The eNOS primers were: forward, 5’-ATC TTC AGC CCC AAA CGG AG-3’) and reverse, 5’-GAT CAG ACC TGG CAG CAA CT-3’). Levels of mRNA were quantified using the SYBR Green Real-Time PCR with the 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). In GeneBank, the sequence of eNOS was NM_000603.5. Primer Premier 5.0 was used to design the primers. PCR was performed in a volume of 10 µl: DNase master mix 5 µl, cDNA 2 µl, ddH2O, forward primer 0.5 µl, and reverse primer 0.5 µl. PCR was performed using: 1) 95°C for 10 min; and 2) 40 cycles of 95°C for 15 s and 60°C for 1 min. The relative expression of eNOS was normalized to that of GAPDH by the 2-∆∆ct method. The primers for GAPDH were: forward, 5’-TGC ACC ACC AAC TGC TTA GC-3’ and reverse, 5’-GGC ATG GAC TGT GGT CAT GAG-3’.

2.13. Western blot The HUVECs were collected and lysed in modified RIPA buffer (100 mM Tris-HCl, 50 mM NaCl, pH 7.4, 1% Nonidet P-40, 10 mg/ml aprotinin, and 10mg/ml leupeptin). The protein supernatants were collected by centrifugation (12,000 g for 10 min at 4°C). Protein concentration was determined by the Lowry method. Western blot was performed according to standard procedures. The following antibodies were used: anti-eNOS (1:800; Abcam, Cambridge, MA, USA); anti-AKT (1:1000; Abcam, Cambridge, MA, USA); anti-PI3K (1:1000; Novus Biologicals, Centennial, CO, USA); or anti-GAPDH (1:1000; Proteintech Group inc., Chicago, IL, USA), and HRP-conjugated goat anti-rabbit or mouse IgG (H+L) (1:5000; MultiSciences,

11

Hangzhou, Zhejiang, China).

2.14. Statistical analysis All values are presented as means ±standard deviation (S.D.). The Student t-test was used to compare two groups. One-way analysis of variance (ANOVA) and Dunnett’s post hoc test was used for multigroup comparisons. P values <0.05 were considered statistically significant. All analyses were performed using SPSS 20.0 (IBM, Armonk, NY, USA).

3. RESULTS 3.1. PlGF decreases IH in vein grafts In order to detect the effect of PlGF release from the hydrogel, ELISA was used to detect the levels of PlGF in the vein grafts at four time points (1, 3, 5, and 7 days after operation). The graft concentration of PlGF was higher in the PlGF group than in the control group on days 1, 3, and 5. The graft concentration of PlGF in the control group was higher than in the Normal group on days 1 and 3 (P<0.01, Fig. 1A). Immunohistochemistry (Supplementary Fig. 2) showed that PlGF could be detected in the tunica adventitia, media, and intima at 1 day after operation, and the staining intensity was higher in the tunica adventitia than in the tunica media, which was higher than in the tunica intima. The expression levels of PlGF in the tunica media/intima decreased with postoperative time. Overall, the results indicated that PlGF could effectively cross the tunica adventitia and reach the tunica media and intima. On the 7th day after operation, IH between the PlGF and control groups showed no significant difference (data not shown). At 14 and 28 days after operation, both groups

12

had uneven intimal thickening (Fig. 1B-E). The number of cells under the endothelium was increased, and most of the nuclei were of the short spindle type. The IH in the PlGF group was less extensive than in the control group, with respect to both thickness (Fig. 1F) and area (Fig. 1G): at 14 days, 8.99±2.04 µm vs. 26.60±8.76 µm (P<0.01, Fig. 1F), and 0.029±0.006 mm2 vs. 0.052±0.007 mm2 (P<0.05, Fig. 1G); and at 28 days, 12.01±1.85 µm vs. 61.68±13.13 µm (P<0.001, Fig. 1F), and 0.049±0.008 mm2 vs. 0.126±0.022 mm2 (P<0.01, Fig. 1G). At 14 days postoperatively, there was no significant difference in the thickness of the tunica media between the PlGF (Fig. 1B) and control (Fig. 1D) groups, (22.88±3.52 µm vs. 25.13±3.84 µm, P=0.359, Fig. 1H). Consistently, there was no significant difference in the area of tunica media between the PlGF (Fig. 1B) and control (Fig. 1D) groups (0.047±0.009 mm2 vs. 0.050±0.008 mm2, P=0.629, Fig. 1I). At 28 days postoperatively, although media thickening was present in the two groups (Fig. 1C and 1E), there was still no significant difference between the PlGF and control groups, either in thickness (Fig. 1H) or area (Fig. 1I): 26.92±6.94 µm vs. 30.04±6.09 µm (P=0.467); 0.056 ± 0.019 mm2 vs. 0.063 ± 0.014 mm2 (P=0.545).

3.2. PlGF promotes the proliferation of microvessels in vein grafts The platelet endothelial cell adhesion molecule-1 (CD31) was used to observe the endothelial cells (Zhou et al., 2016). At 3 days after operation, we observed that the endothelial cells of the rat venous grafts in the two groups were homogeneous or with small differences. According to the percentage of positive cells in the perimeter of the vascular wall, the endothelial integrity was scored as: (1+), <25%; (2+), 25%-49%; (3+), 50%-74%; and (4+), 75%-100%. On day 3, the control group showed 66.7% 1+ and 33.3% 2+, while the PlGF group showed 66.7% 2+ and 33.3% 3+ (P=0.01). On

13

day 7, the control group showed 66.7% 1+ and 33.3% 2+, while the PlGF group showed 50.0% 2+ and 50.0% 3+ (P=0.02). On day 14, the control group showed 50.0% 1+ and 50.0% 2+, while the PlGF group showed 66.7% 2+ and 33.3% 3+ (P=0.03). There was no difference on day 28, both groups having 16.7% 3+ and 83.3% 4+ (P>0.99). (Fig. 2A, Supplementary Table 1). These data suggest that PlGF helped preserve the endothelium to reduce IH. Microvessels were counted by CD31 staining under a 400× microscope (Zhou et al., 2016). On the 3rd day after surgery, the two groups showed almost no angiogenesis in the vein graft wall, but on the 7th day after surgery, there were more microvessels in the PlGF group than in the control group (12.80±2.28 vs. 4.67±2.25, P<0.01). On the 14th day after surgery, there were also more microvessels in the PlGF group than in the control group (16.17±2.48 vs. 6.50±2.88, P<0.01). On the 28th day after surgery, there was no significant difference between the two groups in the amounts of microvessels (9.50±1.87 vs. 7.50±1.87, P=0.094) (Fig. 2B-C). These data indicate that PlGF has the ability to stimulate angiogenesis in the vein graft.

3.3. PlGF increases eNOS and NO levels in vein grafts Immunohistochemistry was used to detect the expression of eNOS in vascular walls. On days 3, 7, and 14 after operation, the content of eNOS in the PlGF group was higher than in the control group (Fig. 3A-B). In the PlGF group, eNOS was mainly expressed in the intima but was also slightly expressed in the adventitia. In the control group, it was mainly expressed in the intima and rarely in the outer layer. On the 28th day post-operation, the levels of eNOS in the two groups tended to be consistent, and eNOS was mainly expressed in the intima of vessels, and little was expressed in the adventitia and tunica media (Fig. 3A). The expression of iNOS was detected in the

14

vascular grafts of the two groups at 3, 7, 14, and 28 days after operation, and its expression was decreased with time, but there was no significant difference in iNOS levels between the two groups at each time point (Fig. 3C-D). NO content was detected by the nitrate reduction assay. On days 1 and 3 after surgery, NO concentration in serum was higher in the PlGF group than in the control group (Fig. 3E). On days 5 and 7 after surgery, there was no significant difference between the two groups (P>0.05). On days 1, 3, and 5 after surgery, NO concentration in the vein graft tissue was higher in the PlGF group than in the control group (P<0.05). At 7 days after surgery, there were no significant differences between the two groups (Fig. 3F).

3.4. PlGF enhances HUVEC survival and cell viability AO-PI staining shows green and red fluorescent cells, indicating viable and dead cells, respectively. With the extension of hypoxia time, the amounts of viable cells gradually decreased, while the amounts of dead cells gradually increased (Fig. 4A). After hypoxia, the survival rate of HUVECS gradually recovered after intervention with different concentrations of PlGF. The survival rate of HUVECS at 10 ng/ml PlGF at 24 h was higher than in the non-PlGF group (81.16±0.88% vs. 76.53±0.75%, P<0.01), and their survival rate at 10 ng/ml PlGF at 48 h was higher than that in the non-PlGF group (84.91±0.52% vs. 81.26±0.54%, P<0.01). The survival rate of HUVECS with 50 ng/ml PlGF at 24 h was higher than with 10 ng/ml PlGF (86.02±1.24% vs. 81.16±0.88%. P<0.01). Their survival rate with 50 ng/ml PlGF at 48 h was higher than with 10 ng/ml PlGF (91.67±0.59% vs. 84.91±0.52%, P<0.01). There were no significant differences between 100 and 50 ng/ml PlGF at 24 and 48 h (Fig. 4B-C). The CCK-8 assay showed that the proliferation of the HUVECs gradually decreased with hypoxia time, and the

15

cell viability decreased from 96.9% to 61.8% after hypoxia for 24 h (Fig. 5A). These results suggest that the effective concentration of PlGF on restoring cell viability was >50 ng/ml, and the optimal treatment time was 48 h (Fig. 5B). These results suggest that PlGF enhances the proliferation and survival of HUVECs.

3.5. PlGF promotes the migration of HUVECs The Transwell assay was used to investigate the influence of PlGF on the migration of HUVECs after hypoxic injury. The results showed that the migration ability of the HUVECs was decreased after hypoxic injury, but migration could be partially recovered after PlGF treatment (Fig. 6A). These results suggest that PlGF enhances the migration ability of HUVECs.

3.6. PlGF promotes the tube formation ability of HUVECs We used different HUVEC models to determine the effect of PlGF on capillary-like tube structure formation ability. The results showed that the tube formation ability of HUVECs under hypoxic conditions was decreased. The three indicators (junction, tubule, and total tubule length) in the hypoxia group were lower than those in the normoxia group (junction, 6.67±1.53 vs. 17.00±2.00, P<0.01; tubule, 14.67±1.53 vs. 34.33±3.06, P<0.01; total tubule length, 117.00±47.43 vs. 3038.67±115.85, P<0.01). After PlGF treatment, the tube formation ability of HUVECs under hypoxic conditions was significantly improved (junction, 24.67±2.08 vs. 6.67±1.53, P<0.01; tubule, 47.67±2.31 vs. 14.67±1.53, P<0.01; total tubule length, 4203.00± 57.28 vs. 1179.00±47.43, P<0.01).

3.7. PlGF promotes NO secretion and NOS expression

16

The content of NO in HUVEC supernatant was detected by the nitrate reductase assay. After hypoxic injury, the concentration of NO in the supernatant was decreased but was obviously increased after PlGF treatment (Fig. 6C). The content of NOS in the supernatant was detected by ELISA. The concentration of NOS in the supernatant was decreased after hypoxic injury but was remarkably increased after PlGF treatment (Fig. 6D). These results suggest that PlGF enhances the expression and activity of NOS in HUVECs.

3.8. PlGF upregulates AKT and the mRNA and protein expression of eNOS in HUVECs after hypoxia qRT-PCR showed that the mRNA expression of eNOS was decreased after hypoxic injury, but was significantly increased after treatment with 50 ng/ml PlGF for 48 h. The mRNA expression of eNOS decreased after the addition of the PI3K inhibitor LY294002 (Fig. 7A). Western blot revealed that PlGF promoted the protein expression of eNOS by up-regulating AKT, and the protein expression levels of AKT and eNOS were decreased after adding the PI3K inhibitor LY294002. These results suggest that PlGF plays a role in upregulating eNOS expression through the PI3K-AKT pathway (Fig. 7B).

4. DISCUSSION The patency of SVG is limited by IH caused by venous endothelial dysfunction. This study aimed to explore the effect of PlGF on the endothelial function of SVG. The results suggest that PlGF is a candidate for the inhibition of IH in SVG after CABG. The effects of PlGF are mediated by the upregulation of the eNOS mRNA and protein through the PI3K/AKT signaling pathway. PlGF promotes the secretion of NO by

17

endothelial cells and reduces the occurrence and development of IH. In the animal experiment, we found that the denudation of the endothelial cells gradually worsened on days 3, 7, and 14 after SVG implantation. In the in vitro experiments, after HUVECs were injured by hypoxia, biological functions such as proliferation, viability, and migration were all decreased. Endothelial cells protect against monocyte adhesion and proliferation of vascular smooth muscle cells (VSMCs), and endothelial cell injury is considered to be the initial event in the development of IH (Bai et al., 2017; Yu et al., 2015). Ehsan et al. (Ehsan et al., 2002) revealed that endothelial cells proliferate and migrate to “reline” the damaged endothelial layer, a process that takes 1-2 weeks, but the newly formed endothelium does not have an intact physiological function (Powell and Gosling, 1998). The remodeling process of neointimal formation and reendothelialization in the early period after CABG is a critical determinant of vessel patency (Harskamp et al., 2013). The results of the present study further support that early injury and dysfunction of endothelial cells trigger IH. Indeed, in the present study, hypoxia decreased HUVEC survival, proliferation, migration, and cell formation ability, supporting the in vivo results of decreased endothelial layer in the vein grafts of the control group. Previous studies of PlGF indicated that PlGF might promote vascular formation by inducing endothelial cell migration and survival and by promoting angiogenesis and arteriogenesis (Park et al., 1994; Skoda et al., 2018; Zhang et al., 2015). Nevertheless, the role and mechanisms of PlGF in inhibiting vein graft IH are still not fully elucidated. Therefore, we used a hydrogel containing PlGF to pretreat the SVG and observed that PlGF significantly reduced the thickening of the intima in the early stage of vein graft transplantation. On the 14th day after SVG transplantation, the intima began to thicken, but was thinner than that of the control group. On the 28th

18

days after operation, the intima of the control group was about five times thicker than that of the PlGF group. In addition, we found that endothelial denudation in the PlGF group was less than in the control group on the 3rd, 7th, and 14th days post-operation. Finally, the outer layer in the PlGF group showed more microangiogenesis and expressed higher levels of endothelial NOS and NO compared with the control group. Those results indicate that PlGF effectively repairs the damaged intima, maintains better structural integrity of the endothelium, and alleviates IH. This is globally supported by Osol et al. (Osol et al., 2008), who showed that PlGF is a potent vasodilator and that it plays a major role in uterine arterial remodeling. In order to further determine the role and mechanism of PlGF in reducing IH, we established HUVEC injury models through hypoxia culture and verified the effect of PlGF on injured HUVEC. The results showed that PlGF restores the proliferation, viability, migration, and tube formation ability in HUVEC after hypoxic injury; that PlGF promotes the secretion of NO and total NOS in HUVECs after hypoxic injury; and that PlGF upregulates the expression of eNOS mRNA and protein in HUVECs after hypoxic injury. These results are supported by previous studies that demonstrated that PlGF increased the concentration of NO in tissues and serum by upregulating eNOS, and that NO stimulated endothelial cell production, migration, and survival, and inhibited endothelial cell apoptosis (Adini et al., 2002; Carmeliet et al., 2001; Powell and Gosling, 1998). eNOS is one of the key enzymes of NO synthesis and is one of the keys to inhibiting IH (Katusic and Austin, 2013; Lundberg et al., 2015; Sessa, 2004). Therefore, interventions such as PlGF could be beneficial for the management of vein grafts after CABG. Recent studies demonstrated that NO exerts vascular protection effects through a variety of biological activities, including promoting endothelial cell migration,

19

proliferation, and tissue reticular formation, followed by the formation of tubes, inhibition of platelet aggregation and leukocyte adhesion, prevention of platelet-derived growth factor release, and reduction of expression of chemotactic protein MCP-1 and multiple surface adhesion molecules to prevent early atherogenesis (Cooke, 2004; Mackenzie et al., 2008). In addition, NO also reduces endothelial permeability, decreases lipoprotein influx into the vessel wall, and prevents low-density lipoprotein oxidation, which might contribute to the antiatherogenic properties of endothelial NOS-derived NO (Cooke, 2004; Mackenzie et al., 2008). Finally, NO has been shown to inhibit VSMC proliferation and migration, thereby preventing the later stages of atherosclerosis (Cooke, 2004; Mackenzie et al., 2008). Based on the combination of these effects, the production of NO by endothelial cells could be considered as the main factor in inhibiting IH (Forstermann and Sessa, 2012; Li and Forstermann, 2000; Papapetropoulos et al., 1997). Based on these findings, we hypothesized that in response to PlGF, the vein graft rapidly forms microvessels, improving oxygen supply in the vein graft and reducing hypoxia, restoring the physiological function of the endothelial cells, and reducing IH. The PI3K/Akt signaling pathway is involved in the regulation of NOS activity and NO production, as well as in angiogenesis (Fulton et al., 1999; Kawasaki et al., 2003). PlGF has been shown to increase the angiogenesis of intestinal endothelial cells via the PI3K/Akt pathway (Zhou et al., 2016). PlGF also increases the proliferation of β cells through the PI3K pathway (Li et al., 2015). In the present study, hypoxic injury decreased the expression of PI3K and NOS, but their expression was partially restored by PlGF. The effects of PlGF were prevented by the PI3K inhibitor LY294002, supporting the involvement of PI3K in the prevention of vein graft IH by PlGF. Nevertheless, PI3K is probably only one of many factors involved in this process, and

20

additional studies are still necessary to determine the exact mechanisms of IH prevention by PlGF. The use of PlGF could possibly become a therapeutic approach to prevent IH after CABG, thus preserving SVG patency and improving patient outcomes. This paper presents vein graft failure as the result of endothelial dysfunction leading to IH and atherosclerosis. This ignores the role of midterm failure occurring before 18 months, which is not well understood and is not affected by atherosclerotic risk factors. This failure is the result of both IH and inadequate positive vein remodeling (i.e., overall increase in vessel size). Additional studies are necessary to determine the causes of graft failure. Importantly, only PlGF was used in the present study, and direct comparison with drugs was not made.

5. CONCLUSION IH remains one of the major obstacles to the long-term patency of SVG. The present study suggests that PlGF is a candidate for the prevention of IH in SVG. PlGF up-regulates eNOS mRNA and protein levels through the PI3K/AKT signaling pathway, promotes the secretion of NO by endothelial cells, and reduces the occurrence and development of IH. These findings contribute to the development of new clinical approaches to enhance SVG function and control IH.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (grant number 81470481).

CONFLICT OF INTEREST

21

The authors declare that there is no conflict of interest regarding the publication of this paper.

22

REFERENCES Adini, A., Kornaga, T., Firoozbakht, F., Benjamin, L.E., 2002. Placental Growth Factor Is a Survival Factor for Tumor Endothelial Cells and Macrophages. Cancer Res 62, 2749-2752. Bai, X., Xi, J., Bi, Y., Zhao, X., Bing, W., Meng, X., Liu, Y., Zhu, Z., Song, G., 2017. TNF-α promotes survival and migration of MSCs under oxidative stress via NF-κB pathway to attenuate intimal hyperplasia in vein grafts. J Cell Mol Med. 21, 2077-2091. Bank, H.L., 1988. Rapid assessment of islet viability with acridine orange and propidium iodide. In Vitro Cell Dev Biol. 24, 266-273. Carmeliet, P., Moons, L., Luttun, A., Vincenti, V., Compernolle, V., De Mol, M., Wu, Y., Bono, F., Devy, L., Beck, H., Scholz, D., Acker, T., DiPalma, T., Dewerchin, M., Noel, A., Stalmans, I., Barra, A., Blacher, S., VandenDriessche, T., Ponten, A., Eriksson, U., Plate, K.H., Foidart, J.M., Schaper, W., Charnock-Jones, D.S., Hicklin, D.J., Herbert, J.M., Collen, D., Persico, M.G., 2001. Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions. Nat Med. 7, 575-583. Cooke, J.P., 2004. The pivotal role of nitric oxide for vascular health. Can J Cardiol. 20 Suppl B, 7B-15B. Davies, M.G., Hagen, P.O., 2011. Pathophysiology of Vein Graft Failure: A Review. Eur J Vasc Endovasc Surg. 42, S19-S29. (Reprinted Article) de Vries, M.R., Quax, P.H.A., 2018. Inflammation in Vein Graft Disease. Front Cardiovasc Med. 5, 3. Dewerchin, M., Carmeliet, P., 2014. Placental growth factor in cancer. Expert Opin

23

Ther Targets. 18, 1339-1353. Ehsan, A., Mann, M.J., Dell'Acqua, G., Tamura, K., Braun-Dullaeus, R., Dzau, V.J., 2002. Endothelial Healing in Vein Grafts: Proliferative Burst Unimpaired by Genetic Therapy of Neointimal Disease. Circulation. 105, 1686-1692. Forstermann, U., Sessa, W.C., 2012. Nitric oxide synthases: regulation and function. Eur Heart J. 33, 829-837. Fulton, D., Gratton, J.P., McCabe, T.J., Fontana, J., Fujio, Y., Walsh, K., Franke, T.F., Papapetropoulos, A., Sessa, W.C., 1999. Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature. 399, 597-601. Gaal, E.I., Tammela, T., Anisimov, A., Marbacher, S., Honkanen, P., Zarkada, G., Leppanen, V.M., Tatlisumak, T., Hernesniemi, J., Niemela, M., Alitalo, K., 2013. Comparison of vascular growth factors in the murine brain reveals placenta growth factor as prime candidate for CNS revascularization. Blood. 122, 658-665. Gaudino, M., Benedetto, U., Fremes, S., Biondi-Zoccai, G., Sedrakyan, A., Puskas, J.D., Angelini, G.D., Buxton, B., Frati, G., Hare, D.L., Hayward, P., Nasso, G., Moat, N., Peric, M., Yoo, K.J., Speziale, G., Girardi, L.N., Taggart, D.P., 2018. Radial-Artery or Saphenous-Vein Grafts in Coronary-Artery Bypass Surgery. N Engl J Med. 378, 2069-2077. Goldman, S., Sethi, G.K., Holman, W., Thai, H., McFalls, E., Ward, H.B., Kelly, R.F., Rhenman, B., Tobler, G.H., Bakaeen, F.G., Huh, J., Soltero, E., Moursi, M., Haime, M., Crittenden, M., Kasirajan, V., Ratliff, M., Pett, S., Irimpen, A., Gunnar, W., Thomas, D., Fremes, S., Moritz, T., Reda, D., Harrison, L., Wagner, T.H., Wang, Y., Planting, L., Miller, M., Rodriguez, Y., Juneman, E., Morrison, D., Pierce, M.K., Kreamer, S., Shih, M.C., Lee, K., 2011. Radial Artery Grafts vs

24

Saphenous Vein Grafts in Coronary Artery Bypass Surgery. JAMA. 305, 167-174. Goldstone, R.N., McCormack, M.C., Khan, S.I., Salinas, H.M., Meppelink, A., Randolph, M.A., Watkins, M.T., Redmond, R.W., Austen, W.G., 2016. Photochemical Tissue Passivation Reduces Vein Graft Intimal Hyperplasia in a Swine Model of Arteriovenous Bypass Grafting. J Am Heart Assoc. 5, pii: e003856. Harskamp, R.E., Lopes, R.D., Baisden, C.E., de Winter, R.J., Alexander, J.H., 2013. Saphenous Vein Graft Failure After Coronary Artery Bypass Surgery. Ann Surg. 257, 824-833. Iwama, H., Uemura, S., Naya, N., Imagawa, K., Takemoto, Y., Asai, O., Onoue, K., Okayama, S., Somekawa, S., Kida, Y., Takeda, Y., Nakatani, K., Takaoka, M., Kawata, H., Horii, M., Nakajima, T., Doi, N., Saito, Y., 2006. Cardiac Expression of Placental Growth Factor Predicts the Improvement of Chronic Phase Left Ventricular Function in Patients With Acute Myocardial Infarction. J Am Coll Cardiol. 47, 1559-1567. Kahraman, N., Yumun, G., Gucu, A., Ozsin, K.K., Taner, T., Sener, E., Goncu, M.T., 2016. Administration of perivascular cyanoacrylate for the prevention of cellular damage in saphenous vein grafts: an experimental model. Cardiovasc J Afr. 27, 159-163. Katusic, Z.S., Austin, S.A., 2013. Endothelial nitric oxide: protector of a healthy mind. Eur Heart J. 35, 888-894. Kawasaki, K., Smith, R.S., Jr., Hsieh, C.M., Sun, J., Chao, J., Liao, J.K., 2003. Activation of the phosphatidylinositol 3-kinase/protein kinase Akt pathway mediates nitric oxide-induced endothelial cell migration and angiogenesis. Mol

25

Cell Biol. 23, 5726-5737. Lardenoye, J.H., de Vries, M.R., Löwik, C.W., Xu, Q., Dhore, C.R., Cleutjens, J.P., van Hinsbergh, V.W., van Bockel, J.H., Quax, P.H., 2002. Accelerated Atherosclerosis and Calcification in Vein Grafts: A Study in APOE*3 Leiden Transgenic Mice. Circ Res. 91, 577-584. Li, F.D., Sexton, K.W., Hocking, K.M., Osgood, M.J., Eagle, S., Cheung-Flynn, J., Brophy, C.M., Komalavilas, P., 2013. Intimal thickness associated with endothelial dysfunction in human vein grafts. J Surg Res. 180, e55-62. Li, H., Forstermann, U., 2000. Nitric oxide in the pathogenesis of vascular disease. J Pathol. 190, 244-254. Li, J., Ying, H., Cai, G., Guo, Q., Chen, L., 2015. Pre-Eclampsia-Associated Reduction in Placental Growth Factor Impaired Beta Cell Proliferation Through PI3k Signalling. Cell Physiol Biochem. 36, 34-43. Lopes, R.D., Mehta, R.H., Hafley, G.E., Williams, J.B., Mack, M.J., Peterson, E.D., Allen, K.B., Harrington, R.A., Gibson, C.M., Califf, R.M., Kouchoukos, N.T., Ferguson, T.B., Alexander, J.H., 2012. Relationship Between Vein Graft Failure and Subsequent Clinical Outcomes After Coronary Artery Bypass Surgery. Circulation. 125, 749-756. Lundberg, J.O., Gladwin, M.T., Weitzberg, E., 2015. Strategies to increase nitric oxide signalling in cardiovascular disease. Nat Rev Drug Discov. 14, 623-641. Luttun, A., Tjwa, M., Moons, L., Wu, Y., Angelillo-Scherrer, A., Liao, F., Nagy, J.A., Hooper, A., Priller, J., De Klerck, B., Compernolle, V., Daci, E., Bohlen, P., Dewerchin, M., Herbert, J.M., Fava, R., Matthys, P., Carmeliet, G., Collen, D., Dvorak, H.F., Hicklin, D.J., Carmeliet, P., 2002. Revascularization of ischemic tissues by PlGF treatment and inhibition of tumor angiogenesis, arthritis and

26

atherosclerosis by anti-Flt1. Nat Med. 8, 831-840. Mackenzie, I.S., Rutherford, D., MacDonald, T.M., 2008. Nitric oxide and cardiovascular effects: new insights in the role of nitric oxide for the management of osteoarthritis. Arthritis Res Ther. 10 Suppl 2, S3. McKavanagh, P., Yanagawa, B., Zawadowski, G., Cheema, A., 2017. Management and Prevention of Saphenous Vein Graft Failure: A Review. Cardiol Ther. 6, 203-223. Meirson, T., Orion, E., Di Mario, C., Webb, C., Patel, N., Channon, K.M., Ben Gal, Y., Taggart, D.P., 2015. Flow patterns in externally stented saphenous vein grafts and development of intimal hyperplasia. J Thorac Cardiovasc Surg. 150, 871-878. Motwani, J.G., Topol, E.J., 1998. Aortocoronary saphenous vein graft disease: pathogenesis, predisposition, and prevention. Circulation. 97, 916-931. Muto, A., Model, L., Ziegler, K., Eghbalieh, S.D., Dardik, A., 2010. Mechanisms of vein graft adaptation to the arterial circulation: insights into the neointimal algorithm and management strategies. Circ J. 74, 1501-1512. Osol, G., Celia, G., Gokina, N., Barron, C., Chien, E., Mandala, M., Luksha, L., Kublickiene, K., 2008. Placental growth factor is a potent vasodilator of rat and human resistance arteries. Am J Physiol Heart Circ Physiol. 294, H1381-1387. Papapetropoulos, A., Garcia-Cardena, G., Madri, J.A., Sessa, W.C., 1997. Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells. J Clin Invest. 100, 3131-3139. Park, J.E., Chen, H.H., Winer, J., Houck, K.A., Ferrara, N., 1994. Placenta growth factor. Potentiation of vascular endothelial growth factor bioactivity, in vitro and in vivo, and high affinity binding to Flt-1 but not to Flk-1/KDR. J Biol Chem.

27

269, 25646-25654. Powell, J.T., Gosling, M., 1998. Molecular and cellular changes in vein grafts: influence of pulsatile stretch. Curr Opin Cardiol. 13, 453-458. Prieto, D., Contreras, C., Sanchez, A., 2014. Endothelial dysfunction, obesity and insulin resistance. Curr Vasc Pharmacol. 12, 412-426. Sabik, J.F., Lytle, B.W., Blackstone, E.H., Houghtaling, P.L., Cosgrove, D.M., 2005. Comparison of Saphenous Vein and Internal Thoracic Artery Graft Patency by Coronary System. Ann Thorac Surg. 79, 544-551. Sessa, W.C., 2004. eNOS at a glance. J Cell Sci. 117, 2427-2429. Skoda, M., Stangret, A., Szukiewicz, D., 2018. Fractalkine and placental growth factor: A duet of inflammation and angiogenesis in cardiovascular disorders. Cytokine Growth Factor Rev. 39, 116-123. Taimeh, Z., Loughran, J., Birks, E.J., Bolli, R., 2013. Vascular endothelial growth factor in heart failure. Nat Rev Cardiol. 10, 519-530. Tousoulis, D., Kampoli, A.M., Tentolouris, C., Papageorgiou, N., Stefanadis, C., 2012. The role of nitric oxide on endothelial function. Curr Vasc Pharmacol. 10, 4-18. Tseng, C.N., Karlof, E., Chang, Y.T., Lengquist, M., Rotzius, P., Berggren, P.O., Hedin, U., Eriksson, E.E., 2014. Contribution of endothelial injury and inflammation in early phase to vein graft failure: the causal factors impact on the development of intimal hyperplasia in murine models. PLoS One. 9, e98904. Valdes, P.J., Diaz, M.A., 2018. Vein Graft Stenosis, StatPearls, Treasure Island (FL). Yu, D., Makkar, G., Strickland, D.K., Blanpied, T.A., Stumpo, D.J., Blackshear, P.J., Sarkar, R., Monahan, T.S., 2015. Myristoylated Alanine‐Rich Protein Kinase Substrate (MARCKS) Regulates Small GTPase Rac1 and Cdc42 Activity and Is a Critical Mediator of Vascular Smooth Muscle Cell Migration in Intimal

28

Hyperplasia Formation. J Am Heart Assoc. 4, e002255. Zhang, J., Chen, A., Wu, Y., Zhao, Q., 2015. Placental Growth Factor Promotes Cardiac Muscle Repair via Enhanced Neovascularization. Cell Physiol Biochem. 36, 947-955. Zhao, Q., Zhu, Y., Xu, Z., Cheng, Z., Mei, J., Chen, X., Wang, X., 2018. Effect of Ticagrelor Plus Aspirin, Ticagrelor Alone, or Aspirin Alone on Saphenous Vein Graft Patency 1 Year After Coronary Artery Bypass Grafting. JAMA. 319, 1677. Zheng, Y., Watanabe, M., Kuraishi, T., Hattori, S., Kai, C., Shibuya, M., 2007. Chimeric VEGF-ENZ7/PlGF Specifically Binding to VEGFR-2 Accelerates Skin Wound Healing via Enhancement of Neovascularization. Arterioscler Thromb Vasc Biol. 27, 503-511. Zhou, Y., Tu, C., Zhao, Y., Liu, H., Zhang, S., 2016. Placental growth factor enhances angiogenesis in human intestinal microvascular endothelial cells via PI3K/Akt pathway: Potential implications of inflammation bowel disease. Biochem Biophys Res Commun. 470, 967-974.

29

FIGURE LEGENDS Fig. 1. Graft concentration of placental growth factor (PlGF), and thickness and area of the tunica intima and tunica media. (A) Graft concentration of PlGF in the two groups. (B-I) Hematoxylin & eosin staining of the vein grafts, including the PlGF group on days 14 (B) and 28 (C) after the operation, and the control group on days 14 (D) and 28 after operation (E). Scale bar: 50 µm. (F-I) Quantification of (B-E). N=6/time point/group. *P<0.05, ** P<0.01, *** P<0.001. Fig. 2. Immunohistochemistry of CD31 and microvessel formation. (A) Examination of the vascular endothelium in vein grafts using CD31 immunohistochemistry. Scale bar: 100 µm. (B) Examination of the microvessel formation in vein grafts using CD31 immunohistochemistry. Representative images of the placental growth factor (PlGF) and control groups on days 3, 7, and 28 after operation. Scale bar: 50 µm. (C) Microvessel density of the vein grafts measured according to (B). N=6/time point/group. **P<0.01. Fig. 3. Immunohistochemistry of endothelial nitric oxide (NO) synthase (eNOS) and inducible nitric oxide synthase (iNOS) in the vein grafts and NO levels. (A) eNOS staining in the vein grafts on days 3, 7, 14, and 28 after surgery. The expression of eNOS is indicated by red asterisk. Scale bar: 10 µm. (B) Quantification of eNOS expression in tunica intima. (C) iNOS staining in the vein grafts on days 3, 7, 14, and 28 after surgery. The expression of iNOS is indicated by red asterisk. Scale bar: 10 µm. (D) Quantification of iNOS expression in tunica intima. (E) Serum NO concentrations. (F) Graft concentrations of NO. N=6/time point/group.*P<0.05; **P<0.01. Fig. 4. Effect of placental growth factor (PlGF) on the survival of human umbilical vascular endothelial cells (HUVECs) upon hypoxia using acridine orange-propidium iodide (AO-PI). (A) The survival of the endothelial cells under

30

hypoxia was examined with AO-PI staining. Shown are the representative images including (a) control group, (b) hypoxia for 3 h, (c) hypoxia for 6 h, (d) hypoxia for 12 h, (e) hypoxia for 18 h and (f) hypoxia for 24 h. Survival rate in (g). (B-C) The survival of endothelial cells under hypoxia treatment of different time together with PlGF treatment of different dose was examined with AO-PI staining. Shown are the representative images in (B), and the statistical results of survival rate in (C). Scale bar: 100µm. N=3. **P<0.01. For (B): **P<0.01 0ng/ml vs. 10 ng/ml; ##P<0.01 10 ng/ml vs. 50 ng/ml. Fig. 5. The effect of hypoxia and placental growth factor (PlGF) on human umbilical vascular endothelial cell (HUVEC) activity using the Cell Counting Kit (CCK)-8 assay. (A) Viability of HUVEC after hypoxia for 6, 12, 18, and 24 h. (B) Compared with the control group, PlGF (50 and 100 ng/ml in the culture medium for >48 h) promotes the recovery of cell vitality. N=3. *P<0.05; **P<0.01. Fig. 6. The effect of placental growth factor (PlGF) on human umbilical vascular endothelial cell (HUVEC) migration, tube formation ability, and nitric oxide (NO) and NO synthase (NOS) levels. (A) Cell migration. a. The control group with normal incubation. b. The hypoxia group. c. The PlGF group with hypoxia incubation and PlGF treatment.Scale bar: 100µm. (B) Tube formation ability. a. The normoxic group with normal oxygen culture. b. The hypoxia control group. c. The PlGF group with hypoxia culture and PlGF treatment. Scale bar: 200 µm. (C) Number of junctions in the normoxic, control, and PlGF groups. (D) Number of tubules in the normoxic, control, and PlGF groups. (E) Tubule length in the normoxic, control, and PlGF groups. (F) NO concentration of the supernatant in each group. (G) NOS concentration of the supernatant in each group. N=3. *P<0.05, **P<0.01. Fig. 7. Effect of placental growth factor (PlGF) on endothelial nitric oxide

31

synthase (eNOS) and PI3K expression in each group. (A) eNOS expression after hypoxic injury or adding the PI3K inhibitor LY294002. (B) Expression of Akt and eNOS proteins after hypoxic injury or adding the PI3K inhibitor LY294002. N=3. *P<0.05; **P<0.01. Supplementary Fig. 1. Surgical procedure of the rat vein graft model. (A) Operation area preparation, the procedure was performed at 16× magnification with an operating microscope. (B) The external jugular vein (1 cm in length) was dissected and ligated. (C) The carotid artery (white arrow) and vagus nerve (black arrow) were dissected. (D) Carotid artery blood flow was blocked by a special vascular clip. (E) The external jugular vein was inserted into the carotid artery, using 6-8 interrupted stitches of 11-0 silk suture per end-to-end anastomosis. (F) Hydrogel containing placental growth factor (PlGF) was dropped onto vein graft adventitia. Supplementary Fig. 2. PlGF immunostaining showing that the placental growth factor (PlGF) could diffuse beyond the adventitia.

32

Not Applicable.