Effect of VEGF/GREDVY Modified Surface on Vascular Cells Behavior

WEI Lai, TAN Jianying, LI Li, WANG Huanran, LIU Sainan, ZENG Zheng, LIU Tao,WANG Jian, CHEN Junying, WENG Yajun

(School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 China)

Abstract: We synthesized B-He/B-GREDVY and immobilized them on avidin-coated surfaces.To examine the immobilization of molecules in the material, the following experiments were performed:fluorescein isothiocyanate (FITC) fluorescence staining, water contact angle and atomic force microscopy(AFM) measurements.Besides, the biological evaluation experiments were also performed, such as platelets adhesion and activation, the culturing of smooth muscle cells (SMC) and endothelial cells (EC).These experimental results show that the modified surfaces could prevent the hyperproliferation of SMC, and promote the proliferation and migration of EC and EPC.Furthermore, the adding of VEGF improved the EC adhesion in a dynamic environment.Generally, it is expected that the modified surfaces could be used to accelerate the formation of the newly endothelial layer for the construction of platforms for coronary artery stent therapy.

Key words: biotin-GREDVY; VEGF; anticoagulation; endothelial cells; endothelial progenitor cells

1 Introduction

Although percutaneous coronary intervention has been the primary therapy for treating coronary artery disease, the treated coronary artery segments inevitably experience significant mechanical trauma[1].Vascular injury may result in neointimal hyperplasia,in-stent restenosis, and acute stent thrombosis.The induced rapid re-endothelializationin vivo(i e,surface functionalization) has been proposed as an alternative approach for overcoming vascular injury.The survival, adhesion, and migration of ECs mainly depend on the interaction with extracellular matrix(ECM) molecules[2].Therefore, several studies have examined the ability of ECM proteins, growth factors[3],and antibodies to serve as surface functionalization molecules of vascular scaffolds.ECM protein molecules can markedly improve endothelializationin vitroandvivo, including fibronectin(Fn)[4], laminin,vitronectin, and collagens[5].As a result, these peptide sequences are more effective in promoting EC adhesion, proliferation, and migration.GREDVY can effectively preserve the biological activity and spatial conformation of REDV by sealing the end region.

The interaction between avidin and biotin is mediated by non-covalent binding, which is solid and difficult to disintegrate and is thus effective for immobilizing bioactive molecules[6].One molecule of avidin can combine with four biotin molecules, which means that more heparin and GREDVY peptides could be immobilized on the surface.

As a widely used anticoagulant, heparin can mediate the inhibition of blood coagulation by ATIII[7].Furthermore, vascular stents coated with heparin have shown excellent anti-thrombotic and anti-intimal.hyperplasia activities[8].The peptide sequence Arg-Glu-Asp-Val (REDV) in the IIICS region of fibronectin can specifically promote the adhesion of ECs[9]and EPCs compared with the adhesion of SMCs and platelets[10],as this sequence can specifically recognize theα4β1 integrin receptor on the cytomembrane of ECs, which is abundant on EC and scarce on SMC[11].REDVmodified surfaces have been confirmed to mediate the adhesion and migration of ECs without promoting the attachment of SMCs[12].However, few studies have examined biotin-groomed peptides on cardiovascular surface modification.One of the main challenges of stents is combining sufficient functional biomolecules to the biomedical material while maintaining the bioactivity of the biomolecules.EPCs homing is similar with the endothelial leukocyte adhesion cascade to some extent.Furthermore, the EPCs respond to hypoxia and up-regulate of the VEGF expression in ischemic tissue and enhance these cells’ homing at sites where endothelial repair is needed.

This study introduced avidin-biotin as a biological amplifier to immobilize bioactive molecules.Biotinylated heparin (B-Hep), biotinylated GREDVY(B-GREDVY) peptide and VEGF were introduced onto the alkali activation surface of titanium through avidin.Material characterization of the B-Hep/B-GREDVY/VEGF modified surface was conducted to examine whether they were successfully immobilized on the surface.Furthermore, hemocompatibility evaluation, such as the adhesion and activation of platelet, was conducted to assess the homeostasis of the microenvironment in damaged vessels.In addition to inducing neovascularization, cytocompatibility evaluation was conducted, which characterized the proliferation of ECs, EPCs, and SMCs.Finally, after comparison, the optimal microenvironment balanced anticoagulation and vascular cell activity was obtained.

2 Experimental

2.1 Materials

Streptavidin (S0170) were purchased from Solarbio Science & Technology Co., Ltd(Beijing, China).Biotin (B4501), VEGF and N-hydroxysuccinimide (NHS, 56480) were purchased from Sigma-Aldrich Shanghai Co.LLC (Shanghai,China).B-GREDVY were obtained from Shanghai Science Peptide Biological Technology Co., Ltd(Shanghai, China).FITC-GREDVY were purchased from Boster Biological Technology Co., Ltd (California,USA).

2.2 Co-immobilization of B-Hep/BGREDVY and VEGF on the modified surface

The biotinylated heparin was prepared in our laboratory following previously described methods[13].Briefly, heparin (3×10-4mol) was added to MES (50 mM) solution.Next, NHS, EDC, and hexyldiamine were added to the above solution sequentially to obtain a mole ratio of –COOH: NHS: EDC: hexyldiamine=2:1:2:2, and the solution was kept for reaction with continuous shaking at 25 ℃ for 4 h.The solution was dialyzed with ultra-pure (UP) water for 24 h.Biotin, NHS and EDC were then added to this solution sequentially to obtain a mole ratio of biotin: NHS:EDC: =3:2:3, mixed with continuous shaking at 25 ℃for 4 h.The solution was dialyzed with UP water for 24 h.After lyophilization, the B-Hep was collected(Fig.1(a)).

Fig.1 Schematic of B-Hep, B-GREDVY and modified surfaces: (a) The structural formula of biotinylated heparin; (b) The structural formula of biotinylated GREDVY; (c) Schematic illustration of modified surfaces

Ti substrate sheets were cut into round disks (Φ10 mm in diameter) and were mirror-polished and washed before use.The cleaned Ti sheets were activated in 2 mol/L NaOH solution at 80 ℃ for 12 h and washed.After activation, the Ti samples were marked as OH,and the OH samples immersed in avidin solution (0.1 mg/mL) were marked as A.B-Hep (5 mg/mL), and B-GREDVY (200 μg/mL) solution were added onto the A samples respectively and marked as AH and AR.For B-Hep (5 mg/mL)/B-GREDVY (200 μg/mL)co-immobilization on the avidin surfaces, the mixing solution was added dropwise onto the A samples(Fig.1(c)) and then marked as HR200.Then, 50 μL VEGF (100 ng/mL) solution was added dropwise onto the HR200 samples and marked as HR200-V.Therefore 5 ng VEGF was immobilized on each sample surface.

2.3 Material characterization of the modified surface

To detect B-REDVY fixed on the surface, the B-GREDVY was connected to FITC fluorescent, and observed them under a fluorescence microscope.The water contact angle on different sample were measured using a goniometer (Krüss GmbH, Germany).A droplet of water was added to the thoroughly dried sample surface, and the computer calculated the angle.To quantitative analysis the releasing of VEGF, HR200-V samples (OH sample as control) were immersed into 1 mL sterile NaCl and were incubated in the 37 ℃with shaking for 12 h, 1d, 3d, 5d, 7 d, 10 d, 14 d and 21 days in sealed plates.After removing the original NaCl of each sample at a specific time, 1 mL sterile and fresh NaCl were added.ELISA was used to detect the content of VEGF in the collected samples solution above using an ELISA kit.The roughness and surface topography of the modified surfaces were characterized by an atomic force microscope (AFM) (Nanowizard II,JPK Instruments, Berlin, Germany) under the tapping mode.The images were analyzed by CSPM Imager software.Arithmeticaverageroughness (Ra) was used to expresssurface roughness.

2.4 Assessment of hemocompatibility

Platelet-rich plasma was centrifuged (at 3 000 rpm for 15 min) using the fresh rabbit whole blood, and then added them onto the modified surfaces and incubated at 25 ºC for 60 min.Then the washed samples were fixed using glutaraldehyde and coated with a layer of gold.The density and morphology of adhered platelets on the modified surfaces were determined by SEM (Philips Quanta 200).

2.5 Evaluation of cytocompatibility

ECs were extracted from the human umbilical vein, and the procedures used for cell culture followed those of a previous study[14].EPCs were extracted from the bone marrow of SD rats; the detailed procedures of the cells extracted and culture are described elsewhere[14].The experimentin vitrowas conducted to determine the cytocompatibility of modified surfaces on the behavior of ECs, SMCs and EPCs, and OH samples were chosen as control.All samples were sterilized under ultraviolet radiation (27 W, wavelength 254 nm) for 30 min before cells were seeded.ECs,SMCs and EPCs were seeded on the modified samples at a density of 5×104cells/mL, and the samples were incubated in a sterile incubator under 5 % CO2at 37℃ for 1 and 3 days, respectively.The cell counting kit-8 (CCK-8) assay was used to determine the number of live cells and their activities.The CCK-8 assay was added to each sample at 1 and 3 days and incubated for 3 h at 37 ℃, and the absorbance was measured at 450 nm.Immunofluorescence staining was conducted to observe the morphology of adherent cells, including the ECs and EPCs.Rhodamine was then added to the sample surfaces, fixed with 2.5 %glutaraldehyde overnight.The morphology of adherent cells on the modified samples was then observed with a fluorescence microscope under dark conditions.

Migration assays for ECs were conducted using the ‘folding method’ to assess the migration ability of ECs[14].Specifically, one side of the sheets was then immersed into the modified suspension and was cultured for 1 day.The other side (the pure Ti surfaces)was immersed in EC suspension at a density of 5×105cell/mL and was cultured at 37 ℃ for 6 h.The modified surfaces were then immersed into the culture medium and incubated for 1 day.During this process, the ECs would migrate from the pure Ti side to the modified side under the induction of B-GREDVY.Finally, the cell adherent surfaces on the modified side were fixed and stained.The distance of EC migration was from the crease line to the migration distance of most cells on the modified side.

A flow chamber was used to apply a continuous flow shear force to EPCs that could simulate the flow state of human blood.The flow experiment was performed at a sterile operating table at 37 ℃ for 4 h.After the flow chamber was connected to the flow system, cells gradually adhered to the modified surfaces with the induction of B-GREDVY.Finally, the number of ECs and EPCs adhered to the modified surfaces were fixed, stained, and observed.

2.6 Statistical analysis

Three uniform samples were conducted for the experiments above.Origin software performed the statistical analyses, and data were expressed as mean with standard deviation (mean±SD).One-way ANOVA in SPSS was used to assess whether groups were significantly different.Differences withp-values below 0.05 (*p< 0.05) were regarded as significant.

3 Results

3.1 Microstructure characterization

FITC fluorescence staining of REDV

Samples without REDV (OH, A, and AH groups)did not produce fluorescence.However, samples with REDV (AR, HR200, HR200-V) showed green fluorescence because of the presence of REDV,demonstrating that REDV was successfully fixed to the sample surfaces (Fig.2(a)).

Fig.2 The surface characterization of the different samples: (a) FITC fluorescence staining of REDV; (b) Water contact angle (mean±SD,n=3, *p<0.05); (c) FTIR of B-Hep, Hep and Biotin; (d) Release of VEGF for HR200-V samples compared to OH samples (mean±SD,n=6); (e) AFM images of the morphology

3.1.1 Water contact angle

The mean contact angle of OH was approximately 7.5° because of the hydrophilic hydroxy (Fig.2(b)).The mean water contact angle on A sample surface was approximately 37°, which could be interpreted as the exposure of hydrophobic groups in avidin.Furthermore, the mean contact angle was significantly decreased after the immobilization of B-Hep,B-GREDVY and VEGF (in the range from 16-20°),as the fixed molecules included hydroxyl, amine,carboxyl, and sulfo groups.

3.1.2 FTIR of B-Hep

FTIR analysis was used to characterize the chemical composition alteration of Hep/VEGF modified surfaces in compared with alkali activated Ti.Fig.2(c) showed the FTIR result of each sample surface.Compared to pristine Ti, a noticeable new peak was observed at 3 386 cm-1on TiOH surface, which was approximate to the hydroxyl (OH) group stretching vibration peak.Besides, two new peaks at about 1 649 and 1 543 cm-1were emerged on avidin modified surface and corresponding to the stretching vibrations of amideI(C O) and II (C N, N H) bond of avidin.Therefore, we can infer that the avidin were assembled to the alkaline surfaces successfully.Heparin showed a strong characteristic peak at 3 341cm-1, biotin showed a robust characteristic peak at 2 943 cm-1.The FTIR of B-Hep showed solid characteristic peaks at 3 341 and 2 943 cm-1, which may be speculated that B-Hep synthesized successfully.Moreover, the FTIR of B-Hep showed firm characteristic peaks at 1 693-1-625 cm-1and 1303-1228 cm-1, which were ascribed to the C=C bond, and C-S-C bond, respectively.

3.1.3 Release of VEGF

Fig.2(d) showed the release result of VEGF, and OH samples were set as control.The VEGF release exhibited approximate linear distribution from 12 hours to 21 days, which suggested that the release of VEGF was uniform.The cumulative release of VEGF was released 0.9 ng for 21 days.

3.1.4 AFM

The AFM images that show the morphology at the nanometer scale of the modified sample surfaces are shown in Fig.2(e).After NaOH was etched, the surface became rougher, and many granules appeared(Ra=15.9±0.7 nm).After the biological molecules were fixed, the sample surface appeared to be smoother than the OH sample, which might stem from the fact that the biological molecules filled the pores.The surface roughness continued to decrease, and the surface roughness of the modified surfaces was between 10-13 nm.The surface roughness of the HR-200-V was 12.1±0.4 nm, which was the same with the AR and AH samples.The experiment results suggested that the biological molecules were introduced onto the alkaliactivated surface from the side.

3.2 Hemocompatibility evaluation—platelet adhesion activation

The number and the activated state of adherent platelets on the modified surfaces are the leading indicators for evaluating hemocompatibility.Fig.3(a)shows the theoretical model by which the adhesion of platelets is suppressed on the modified surfaces.In general, the morphologies of platelets are interrelated to their activated level, including their typical shape:round, dendritic, spreading dendritic, or spreading or fully spreading (Fig.3(b)).SEM was conducted to determine the morphologies of platelets on modified surfaces (Fig.3(c)), and Fig.3(d) showed the number of platelets adherent to the surface.After alkali-activated,the adherent platelets mainly exhibited spreading dendritic shape, indicating severe activation and poor hemocompatibility.For A and AR samples, the morphologies of adherent platelets indicated that the platelets activated partly.However, after B-Hep were immobilized, the adhered platelet density significantly decreased, and the morphologies of platelets became round.Thus, AH, HR200 and HR200-V could inhibit the adhesion of platelets to a large degree.The number of adherent platelets was significantly different between the OH and HR groups (Fig.3(d)).

Fig.3 Results of the platelet adhesion assessment: (a) Schematic illustration of modified surfaces under platelets; (b) Platelet adhesion behavior: round, dendritic, spreading dendritic, spreading and fully spreading; (c) SEM image of adherent platelets on modified surfaces; (d) The number of adherent platelets on modified surfaces (mean±SD, n=3, *p<0.05)

3.3 Static culture of vascular cells

3.3.1 EC adhesion and proliferation

The REDV peptide-modified surface in damaged vessels can promote the proliferation and migration of ECs and thus contribute to maintaining the homeostasis of the microenvironment (Fig.4(a)).REDV can selectively promote the growth of ECs because REDV on the IIICS CS-5 site could specifically combine with the α4β1 receptor on ECs[12](Fig.4(b)).The model pattern of fibronectin dimer is shown in Fig.4(c), and one molecule of GREDVY has three carboxyl groups.The ECs typically had cobblestonelike morphology, and the conditions were satisfactory on day 1(Fig.4(d)).The number of adhesion cells decreased, and the condition of cells on the alkaliactivated surface was poor due to the presence of avidin with a positive charge.For groups that immobilized B-Hep alone (AH group), the amounts of ECs adhered decreased, and resulting the inhibition of heparin.The number of cells attached was significantly increased on the surface with the AR, HR200 and HR200-V, and the cells had superior morphology and conditions compared with the OH surface because of the presence of B-GREDVY after culturing for 1 day(Figs.4(d)-4(e)).At 3 days, the number of ECs on the OH surface decreased, whereas the number of ECs increased in the HR200 and HR200-V groups (Fig.4(f)).The proliferation ratio was calculated by the CCK-8 value at day 3 normalized to day 1[15].The proliferation rate from 1 to 3 days indicated that the growth of ECs was negative without bioactive molecules on the OH group.The other samples conferred higher proliferation activity of ECs.Combined with the number of adherent cells, the HR200 and HR200-V groups could strongly promote the attachment and proliferation of ECs(Figs.4(f) -4(g)).

Fig.4 EC compatibility assessment: (a) Schematic illustration of modified surfaces under ECs and SMCs; (b) The connection between fibronectin and ECs; (c) The model pattern of fibronectin dimer; (d) Immunofluorescence staining results of ECs after 1 day of culture;(e) The density of ECs after 1 and 3 days of culturing (mean±SD, n=3, *p<0.05); (f) Immunofluorescence staining results of ECs after 3 days of culturing; (g) The proliferation ratio of ECs (mean±SD, n=3, *p<0.05).The proliferation ratio was calculated by the CCK-8 value at day 3 normalized to day 1

3.3.2 SMC adhesion and proliferation

The REDV peptide-modified surface in damaged vessels can selectively inhibit the adhesion of SMCs and thus contribute to maintaining the homeostasis of the microenvironment (Fig.5(a)).The number of SMCs was markedly lower in the HR200 and HR200-V groups than the A surface after culture for 1 and 3 days, which suggested that the biological viability of the SMCs was inhibited in the HR200 and HR200-V groups (Figs.5(b)-5(c)).After 3 days of culture, the SMCs showed the same pattern (Fig.5(b)).The SMCs had an elongated spindle morphology on the OH group, whereas, the SMCs had a shrunken morphology in the HR groups.At 3 days, the number of SMCs on the A surface increased, whereas the number of SMCs decreased in the HR200 and HR200-V groups(Fig.5(d)).For the proliferation ratio of SMCs, all samples have no significant differences (Fig.5(e)).

Fig.5 SMC compatibility assessment: (a) Schematic illustration of the modified surfaces under ECs and SMCs; (b) Immunofluorescence staining of SMCs after culturing for 1 day; (c) Immunofluorescence staining results of SMCs after 3 days of culturing; (d) The density of SMCs after 1 and 3 days of culturing (mean±SD, n=3, *p<0.05); (e) The proliferation ratio of SMCs (all samples have no significant differences.The proliferation ratio was calculated by the CCK-8 value at day 3 normalized to day 1)

3.4 Dynamic culture of vascular cells

3.4.1 EC migration assays

The migration of ECs was another significant indicator for evaluating the endothelialization ability.We used the ‘crease’ method to evaluate the migration distance, and the schematic diagram is shown in Fig.6(a).ECs could migrate from the unmodified part,on the left side of the figure (Ti point) to the modified part, which was on the right side of the figure.Fig.6(b)showed that the migration distance varies widely from sample to sample.After culturing for 1 day, the migration distance of OH was the shortest, and the AR, HR200 and HR200-V groups have increased much in migration distances.For the modified group,the migration distance of the HR200 and HR200-V groups were significantly higher than that of the others(1 600 μm) (Fig.6(c)), from which a conclusion could be drawn that the more suitable the morphology and activity of the ECs, the stronger the migration ability of the ECs was.

Fig.6 ECs migration on the surfaces of the modified samples: (a) Graphic illustration of EC migration; (b) EC migration on the surfaces of the modified samples; (c) Migration distance of different samples (mean±SD, n=3, *p<0.05)

3.4.2 EC culture underflow

Under the flow-simulated body condition, there were few adherent cells for the OH group with alkali activation (Fig.7(a)).The number of ECs increased due to the electrostatic interaction of avidin for the A group.Because of the addition of heparin, the AH group showed a low affinity for ECs.However, the introduction of GREDVY could specifically promote the adhesion and capture of ECs.The AR and HR200 groups showed the most adherent cells, and most cells had a spreading morphology after culturing for 4 h(Fig.7(b)).Furthermore, for HR200-V, the introduction of VEGF could significantly improve the number of adherent ECs.

Fig.7 Cytocompatibility assessment underflow: (a) Immunofluorescence staining results of ECs after 4 h of culturing underflow; (b) The density of ECs after 4 h of culturing underflow (mean±SD, n=3, *p<0.05); (c)Immunofluorescence staining results of EPCs after 4 h of culturing underflow; (d) The density of EPCs after 4 h of culturing underflow (mean±SD, n=3, *p<0.05)

3.4.3 EPC culture underflow

After culturing for 4 h, the adherent amounts of EPCs on the OH surface were comparatively small and had poor spreading morphology (Figs.7(c)-7(d)).Compared with the OH group, the AR, HR200 and HR200-V groups had more adherent EPCs and better spreading morphology, especially the HR200-V group,which demonstrated that GREDVY and VEGF had excellent abilities to capture and promote the growth of EPCs.

4 Discussion

Synergetic effects of heparin and GREDVY on the balance of anticoagulation and endothelization

Implantable cardiovascular devices such as stents have become an effective treatment tool for coronary artery disease.However, thrombosis and stenosis are the main obstacles that limit the long-term effectiveness of the stent.Therefore, the construction of an intact endothelium on the inner lumen of stents has been recognized as an ideal approach to solving the current problems of the stent[16].Coronary artery stents modified through specific biomolecules for selective restrain thrombosis formation and promoting and inducing endothelialization have become a more effective research hotspot.The B-Hep were fixed on the surfaces to promote anticoagulation and antihyperplasia.SEM images indicated that heparin strongly inhibited platelet adhesion (Fig.8).

Fig.8 The effect of the modified microenvironment on platelets and ECs

Furthermore, GREDVY can also suppress platelet adhesion[17].The fluorescent images in Fig.7 revealed that the modified microenvironment could inhibit the growth of SMCs.Because of its unique pentasaccharide ring structure and sulfonate acid group, heparin can inhibit the growth of SMCs[18].From the comparison of platelet adhesion and the adhesion of vascular cells, a conclusion can be drawn that the immobilization of biomolecules may affect the anticoagulant performance to a certain extent(Fig.8).However, the introduction of biomolecules significantly enhanced the cell compatibility of the microenvironment.The samples in this manuscript could promote EC proliferation, migration, and achieve dynamic capture of EC and EPC.The problem of drugeluting stents is the lack of factors that could promote EC growth, resulting in delayed endothelialization.In this manuscript, the anticoagulant heparin is used,the REDV which could promote EC adhesion, and the VEGF that can capture EPC are also used.Therefore,the samples in this manuscript could achieve rapid endothelialization to the greatest extent.From Fig.1(c),we could see that the samples of ‘OH’ and ‘A, AH, AR’showed the difference of substrate and the single drug.The OH sample showed bad cytocompatibility and lots of platelet adhesion.The addition of avidin and B-GREDVY improved cytocompatibility, and B-hep added improved blood compatibility.The above samples excluded chemistry drug and substrate differences.Moreover, the adding of biomolecules significantly increased the adhesion of cells and inhibited platelet adhesion compared to those above samples.The “HR200 and HR200-V” showed better cytocompatibility and blood compatibility.Therefore,the improved parts contributed to the adding of B-GREDVY and VEGF.Thus, synergetic effects of heparin and GREDVY on the balance of anticoagulation and endothelization could be built.This study had suggested that the modified microenvironment could also trap the vascular cells and inhibit the formation of platelets simultaneously.Plouffeet al[19]suggested that the loading of REDV peptides under microfluidic shear flow could capture the most significant number of ECs compared with other kinds of cells.Seetoet al[20]found that REDV could promote the dynamic adhesion of EPCs after they connected with PEG.Previous research has suggested that the concentrations of REDV show high specificity for vascular cells[21].Co-culture of the modified layer revealed that GREDVY had a natural affinity with endothelial cells.From the above results,in terms of promoting ECs adhesion and EPCs capture,the HR200 group showed better abilities than AH and AR groups.Therefore, the presence of B-Hep or GREDVY, may play a positive role in the biological activities of another molecule.

5 Conclusions

In this study, we introduced bioactive molecules in B-Hep and B-GREDVY through biotin-avidin systems,which permitted the number of immobilized molecules to be amplified.After B-Hep and B-GREDVY were immobilized on the OH surface, the hemocompatibility of samples improved substantially compared with the OH surface.In addition, heparin could prevent platelet adhesion and the growth of SMCs, and GREDVY could facilitate the attachment and proliferation of ECs and EPCs.B-Hep and GREDVY may play synergetic effects and promote the biological function of another molecule.In addition, the introduction of VEGF further improved the ability to capture ECs and EPCs in a dynamic environment and further accelerated the formation of the newly endothelial layer for the microenvironment.Compared to other groups,HR200-V showed the best results in cytocompatibility and hemocompatibility.The results of this study could be applied to the design of surface-modified layers immobilized on coronary artery stents to improve reendothelialization.

Conflict of interest

All authors declare that there are no competing interests.