Bioabsorbable zinc ion induced biphasic cellular responses in vascular smooth muscle cells

Bioabsorbable metal zinc (Zn) is a promising new generation of implantable scaffold for cardiovascular and orthopedic applications. In cardiovascular stent applications, zinc ion (Zn2+) will be gradually released into the surrounding vascular tissues from such Zn-containing scaffolds after implantation. However, the interactions between vascular cells and Zn2+ are still largely unknown. We explored the short-term effects of extracellular Zn2+ on human smooth muscle cells (SMCs) up to 24 h, and an interesting biphasic effect of Zn2+ was observed. Lower concentrations (<80 μM) of Zn2+ had no adverse effects on cell viability but promoted cell adhesion, cell spreading, cell proliferation, cell migration, and enhanced the expression of F-actin and vinculin. Cells treated with such lower concentrations of Zn2+ displayed an elongated shape compared to controls without any treatment. In contrast, cells treated with higher Zn2+ concentrations (80–120 μM) had opposite cellular responses and behaviors. Gene expression profiles revealed that the most affected functional genes were related to angiogenesis, inflammation, cell adhesion, vessel tone, and platelet aggregation. Results indicated that Zn has interesting concentration-dependent biphasic effects on SMCs with low concentrations being beneficial to cellular functions.

the corroding Zn implant. After 6.5 months, there was no neointimal tissue thickness progression. No obvious SMCs growth around the implant was observed during the entire course of experiment. It demonstrated that Zn might suppress the SMCs activities 16 which is closely related to restenosis. In vitro study showed that pure Zn had lower corrosion rate, better hemocompatibility, and low cytotoxicity for cardiovascular stent application, compared to high purity Mg 17 . Zn alloying with other elements, such as Mg, Ca, Sr, and Mn were also explored regarding mechanical strength, corrosion resistance, biocompatibility, and hemocompatibility with encouraging outcome 11,[18][19][20] . These studies demonstrated the potential of Zn as promising material for cardiovascular stent application.
Restenosis is one of the main problems reported in stent application 21 . SMCs migration, proliferation and excessive extracellular matrix (ECM) deposition, are responsible for restenosis after stent implantation 21 . Therefore, understanding of how Zn 2+ affect the cellular behaviors of SMCs, especially cell proliferation and cell migration, could provide useful information on the mechanism of restenosis for Zn-based stent. In this study, we evaluated the effects of extracellular Zn 2+ on cellular behaviors of SMCs in a short-term fashion up to 24 h, including cell viability, cell proliferation, cell adhesion, cell spreading, cell migration, cytoskeleton reorganization and cell morphology. Moreover, we also explored the differential gene expression changes of SMCs treated with Zn 2+ , which is the root cause of the cellular behavior changes.

Materials and Methods
Zn 2+ solutions preparation. ZnCl  Cell culture. Human aorta smooth muscle cell (HASMC, ScienCell, US) was expanded in 75 cm 2 culture flask (Falcon, BD Biosciences, US). The culture medium was SMCM with 2% fetal bovine serum (FBS, ScienCell, US), 1% SMC growth supplement (SMCGS, ScienCell, US) and 1% penicillin/streptomycin solution (P/S, ScienCell, US). When cells reached 90% confluence, they were washed by Dulbecco's phosphate-buffered saline (DPBS, Life technologies, US) and then detached by trypsin/EDTA solution (Life technologies, US). After detachment, SMCM was added and the cell solution was centrifuged (Sorvall Biofuge Stratos, Thermo Electron Corporation, US). The supernatant was removed and cell pellet was resuspended by SMCM. The cell solution was mixed with trypan blue stain (Life technologies, US) and cell number was counted by hemocytometer (Bright-Line, Hausser Scientific, US) under microscope (EVOS FL Cell Imaging System, AMG, US). Then cell solution was diluted into different densities for the following tests.
Cell viability test. Cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Life technologies, US). SMCs were seeded into a 96-well plate (Falcon, Corning, US) at density of 5,000 cells/well 100 μL and incubated for 24 h. Cell medium was replaced by different Zn 2+ solutions and incubated for 24 h. After that, solutions were replaced by 100 μL fresh medium supplemented with 10 μL of 12 mM MTT stock solution and incubated for 4 h. Then 100 μL SDS-HCl solutions were added to each well and incubated for another 4 h. The absorbance was measured by microplate reader (Molecular Devices, US) at 570 nm. SMCM with and without cells were used as positive and negative control, respectively. Cell viability was determined as below: were taken by microscope and analyzed by Image J (NIH, US). At least 10 different fields were used for calculating attached cell density and cell retention ratio. Statistical analyses. All data were presented as mean ± standard deviation and at least three replicates were used in each test for each concentration group. Student's t-test was used in statistical analyses (Prism 5, GraphPad Software, US) and p < 0.05 was considered significant.

Results
High concentration of Zn 2+ inhibited cell viability. When below 80 μM, Zn 2+ had no adverse effects on cell viability but above 100 μM, Zn 2+ inhibited cell viability and significance was observed when concentration reached 120 μM (p < 0.001) (Fig. 1). In addition, pH in diluted Zn 2+ solutions had no obvious changes (data not shown).
Scientific RepoRts | 6:26661 | DOI: 10.1038/srep26661 Zn 2+ had biphasic effects on cell proliferation. As shown in Fig. 2, Zn 2+ had a bell-shape biphasic effect on cell proliferation. Zn 2+ promoted cell proliferation with increasing concentration and reached maximum around 80 μM, and then proliferation started to decrease with further increasing of Zn 2+ concentration. For all concentrations up to 110 μM, the proliferation profile remained above base line (100%). However, Zn 2+ inhibited cell proliferation significantly when it was above 120 μM (p < 0.001).
Zn 2+ altered cell adhesion and cell adhesion strength in dose-dependent manners. Cell adhesion was in a Zn 2+ concentration-dependent manner. Cells were allowed 2 h to attach. Zn 2+ increased cell adhesion density when below 40 μM while inhibited cell adhesion when above 40 μM (Fig. 3a). Interestingly, Zn 2+ had an opposite effect on cell retention compared to cell adhesion. After centrifuge, lower percentage of cells remained attached when treated with lower concentration of Zn 2+ (0-40 μM) while higher percentage of cells remained attached for higher concentration (80-120 μM) (Fig. 3b).
Zn 2+ induced biphasic changes on cell spreading. Cell spreading was examined with one typical low (40 μM) and one typical high (120 μM) concentrations of Zn 2+ for up to 8 h (Fig. 4). At 40 μM, Zn 2+ promoted cell spreading and cells had a larger cell area and perimeter. In contrast, at 120 μM, Zn 2+ inhibited cell spreading and cells tended to have smaller cell area and perimeter. For all experimental groups, cell spreading reached a plateau phase after 8 h.
Zn 2+ had biphasic effects on cell migration. Similar to cell proliferation data, cell migration rate had a bell-shape relationship with Zn 2+ concentrations (Fig. 5). Zn 2+ increased cell migration rate when below 80 μM while decreased cell migration rate significantly when above 100 μM (p < 0.001).
Effects of Zn 2+ on cytoskeleton reorganization and cell morphology. Similar to previous cell spreading test, cytoskeleton reorganization and cell morphology were examined with one typical low (40 μM) and one typical high (120 μM) concentrations of Zn 2+ . The representative images were shown in Fig. 6a. Compared to control, cells treated with 40 μM Zn 2+ had larger cell area and perimeter (Fig. 6b,c). In contrast, cells treated with  120 μM Zn 2+ tended to have smaller area and perimeter (Fig. 6b,c). Cell morphology was characterized by aspect ratio and circularity. Cells were likely to display an elongated shape when treated with 40 μM Zn 2+ . Cells displayed a more round shape when treated with 120 μM Zn 2+ compared to controls without any treatment (Fig. 6d,e). Actin and vinculin expression were enhanced at 40 μM Zn 2+ but inhibited at 120 μM Zn 2+ (Fig. 6f,g).

Discussion
Zn is a promising biodegradable metal for cardiovascular stent applications due to its good mechanical and corrosion properties as well as biocompatibility. However, there is very limited study on how Zn may affect the vascular cell behaviors, especially SMCs which is the major player in stent restenosis and thrombogenesis. Endothelial cells and SMCs are the main cellular components of the vessel tissue. They play important roles in growth and maintenance of physiological functions and normal structure of vessel wall, as well as in vascular diseases 22,23 . In cardiovascular stent application, endothelial cell proliferation and migration affect re-endothelialization, which is an important factor to evaluate the overall performance of stent materials 4,24 . After stent implantation, rapid re-endothelialization on the stent surface are essential in preventing intimal thickening and vascular thrombosis 25 . In contrast, proliferation and migration of SMCs and extracellular matrix deposition contribute to restenosis 26 . Such different cellular behaviors and outcome from endothelial cell and SMCs led us to hypothesize that Zn ion may have different effects on these two types of vascular cells. Here, we explored the interesting effects of Zn ion on SMCs as a continuous investigation of Zn and vascular interactions 25 .
Understanding the cellular behaviors of endothelial cells and SMCs exposed to surrounding microenvironment are critical in stent application 25,27 . Intense policy regulation on implantable medical device would require such information to be fully disclosed for any Zn-based device. Therefore, it is essential to understand the interactions between the local vascular cells and Zn ion at the cellular and molecular levels. Here, we reported for the first time that how Zn ion changes the cellular behaviors of SMCs in vitro in a short term.
Zn 2+ altered SMCs viability and proliferation in a dose-dependent manner. Lower concentrations of Zn 2+ had no obvious effects on viability up to 80 μM Zn 2+ but significantly inhibited cell viability and proliferation at 120 μM. This is in line with a previous study that Zn 2+ had no effect on cell viability of human airway SMCs if below ~77 μM and decreased cell viability significantly if above ~115 μM 28 . However, different SMCs from different species may respond differently. For example, at ~38 μM, Zn 2+ significantly inhibited proliferation of SMCs from carotid artery of Wistar rat. In a carotid artery injury model, injection of 5 mg/kg ZnCl 2 for 14 days significantly inhibited neointimal formation and increased lumen area slightly 29 . SMCs from tracheas and bronchi of Brown Norway rat had a significantly higher proliferation with a 3-24 μM concentration range of Zn 2+ , and cell proliferation was significantly inhibited by Zn 2+ at 96 μM 30 . In addition, SMCs from different tissue origin in the same species may have different responses. Human SMCs from benign prostatic hyperplasia had higher Zn 2+ tolerance compared to human coronary SMCs. Zn 2+ promoted prostatic SMCs proliferation with a range of 50-250 μM while significantly inhibited their proliferation when above 250 μM. The underline molecular signaling pathway for such biphasic effects of Zn on SMCs could be a complex. The activation of mitogen-activated protein kinases (MAPKs) and phosphoinositol 3-kinase (PI3K) pathways by Zn 2+ may count for the enhanced cell proliferation including human bronchial epithelial cell 31 and Swiss 3T3 cell 32 . MAPK pathway was also involved in a biphasic relationship between Zn 2+ and HT-29 colorectal cancer cell growth 33 . Treatment with 10 μM Zn 2+ activated ERK transiently and induced cell cycle regulator cyclin D1 while 100 μM of Zn 2+ induced prolonged ERK activities and the increase of cyclin D1 and p21 Cip/WAF1 33 .
Cell adhesion and spreading were also largely dependent on Zn 2+ concentrations. The threshold concentration of Zn 2+ for promoting or inhibiting SMCs adhesion was ~40 μM in this study. The cell-substrate adhesion involves proteins in ECM, transmembrane receptors and cytoskeleton. Transmembrane receptors and cytoskeleton serve as local anchorage and link cells to ECM 34 . Of the genes responsible for SMC-substrate adhesion examined, only ITGB3 was significantly inhibited at 120 μM of Zn 2+ . Additionally, F-actin and vinculin expression were enhanced significantly at 40 μM Zn 2+ while decreased at 120 μM. Previous study revealed that vinculin could promote cell spreading by stabilizing focal adhesions and transferring mechanical stresses that drive cytoskeletal remodeling 35 . This result was consistent with our observation that overexpressed vinculin was accompanied by the promoted cell spreading at 40 μM Zn 2+ . Moreover, cell spreading is tightly related to cell proliferation. Cell spreading is often accompanied by changes in the structure and composition of the cytoskeleton and distinctive changes in cell behaviors, such as growth and dedifferentiation 36 . Study showed that restriction of SMC spreading in one direction changed cell morphology and decreased cell proliferation 37 . When cell spreading was confined within a small cell area ranging 300-500 μm 2 on a micropatterned matrix, cell proliferation decreased, probably because of a limited cell spreading 38 . In consistent with previous studies, cell area was within a small range of 200-400 μm 2 when cell spreading reached a plateau phase and decreased cell proliferation was accompanied with decreased cell area and perimeter in this study. The inhibitory effects of confined cell spreading on cell growth was probably because the limited spreading reduced the intracellular pH 39 . Zn 2+ could activate Na + /H + exchanger 1 (NHE1) by binding to an extracellular zinc-sensing receptor (ZnR) and triggering the release of calcium ion (Ca 2+ ) that subsequently activates extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and MAPK pathway 40 . The confined SMC spreading accompanied with decreased cell proliferation at higher concentration of Zn 2+ was probably due to the deactivation of NHE1 by higher concentration of Zn 2+ , leading to a slight decrease in intracellular pH and thereby inhibiting cell growth.
Cell morphology changes requires the transfer of mechanical forces between the cytoskeleton and ECM as well as alterations in cytoskeletal organization 35 . It is well recognized that cell morphology is tightly coupled to DNA synthesis and cell growth 41 . We found that lower concentrations of Zn 2+ promoted DNA synthesis and converted SMC from a relatively round shape to a spindle shape. The relatively elongated shape resembles the morphology of SMC in vivo under physiological condition 42 , which might indicate the beneficial effects of lower concentrations of Zn 2+ . SMC migration, proliferation and excessive ECM deposition contributes to restenosis after stent implantation 21 . In this study, we found lower concentrations of Zn 2+ promoted cell migration while higher concentrations of Zn 2+ inhibited it. To remain motile, a cell must maintain a low level of adhesion to the extracellular matrix to allow traction 43 . Centrifugation assay showed that lower concentrations of Zn 2+ had a lower cell retention ratio and high concentrations of Zn 2+ increased cell retention ratio. These data indicated that cells treated with lower concentrations of Zn 2+ had a lower level of adhesion strength with less firm attachment. This observation might explain the corresponding biphasic effects of Zn 2+ on cell migration.
Finally, we investigated the gene expression profiles of SMCs because they are the ultimate root causes for any observed cellular behavior in most cases. Gene expression data revealed that the most affected functions were angiogenesis, inflammation, cell adhesion (cell-cell adhesion and cell-substrate adhesion), vessel tone, and platelet aggregation. For on particular cell function change, there might be several genes involved in with different levels of regulation. For example, FLT1 gene, encoding vascular endothelial growth factor receptor 1, was significantly down-regulated at 120 μM of Zn 2+ , whereas another gene VEGFA, encoding vascular endothelial cell growth factor A, was significantly up-regulated. These two genes were both involved in angiogenesis but regulated differentially by the same concentration of Zn 2+ . The overall phenotype, therefore, is a combination of different genes regulated at different levels.
Despite some similarities in trend of cellular responses from endothelial cells and SMCs to Zn ions, there are significant differences. Although both endothelial cells and SMCs have biphasic cellular behaviors with exposure to Zn ions, one of the most notable differences was the threshold concentration for specific cellular behavior 25 . Within the same concentration range, it is of great interest to compare the tolerance of endothelial cells and SMCs to Zn ions for cardiovascular stent application. We found that endothelial cells had higher tolerance of Zn ions than SMCs within 0-140 μM even though there were some variances for cell proliferation, which is consistent with recent studies 44,45 . Since endothelial cells directly contact with stent surface, a higher Zn tolerance could be beneficial for re-endothelialization with minimal chance of restenosis. In vivo study also showed that progression of neointimal tissue was checked and the activities of SMCs might be suppressed by the corrosion products of Zn 16 . It was likely that the concentration of Zn ions around the implant may exceed the cytotoxicity threshold concentration for SMCs, which provided a reasonable explanation for the inhibition of SMCs by Zn implants. Taken together, in the future designing of Zn alloys for stents, it would be beneficial to control the local concentration of Zn ion from implant degradation above cytotoxicity threshold for SMCs while below that of endothelial cells while the corrosion rate and mechanical properties should still match the healing phases of injured vessels 46 .
In conclusion, lower concentrations (<80 μM) of Zn 2+ had no adverse effects or beneficial effects on SMC adhesion, spreading, viability, proliferation, and migration. Moreover, lower concentrations of Zn 2+ enhanced the expression of actin and vinculin and cells displayed an elongated shape. Gene expression profiles showed that significantly affected genes were related to angiogenesis, inflammation, cell adhesion, vessel tone, and platelet aggregation. Results revealed that the biocompatibility and cellular behaviors were tightly related to Zn 2+ concentrations. Therefore, this study could provide useful information for a better stent design. A controlled release of Zn ion from Zn-based stent through controlled degradation pace could regulate SMC behaviors and the occurrence of restenosis.