Magnesium used in bioabsorbable stents controls smooth muscle cell proliferation and stimulates endothelial cells in vitro

Katrin Sternberg,1* Matthias Gratz,2* Kathleen Koeck,2 Joerg Mostertz,3 Robert Begunk,2 Marian Loebler,1 Beatrice Semmling,4 Anne Seidlitz,4 Petra Hildebrandt,3 Georg Homuth,3 Niels Grabow,1 Conny Tuemmler,5 Werner Weitschies,4 Klaus-Peter Schmitz,1 Heyo K. Kroemer2
1 2

University of Rostock, Medical Faculty, Institute for Biomedical Engineering, Rostock, Germany Ernst-Moritz-Arndt University, Medical Faculty, Institute of Pharmacology, Greifswald, Germany 3 Ernst-Moritz-Arndt University, Interfaculty Institute for Genetics and Functional Genomics, Junior Research Group Transcriptomics/Functional Genomics, Greifswald, Germany 4 Ernst-Moritz-Arndt University, Faculty of Mathematics and Natural Sciences, Institute of Pharmacy, Greifswald, Germany 5 University of Rostock, Faculty of Mathematics and Natural Sciences, Institute for Biological Sciences, Rostock, Germany Received 28 May 2010; revised 24 March 2011; accepted 2 June 2011 Published online 24 November 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.31918 Abstract: Magnesium-based bioabsorbable cardiovascular stents have been developed to overcome limitations of permanent metallic stents, such as late stent thrombosis. During stent degradation, endothelial and smooth muscle cells will be exposed to locally high magnesium concentrations with yet unknown physiological consequences. Here, we investigated the effects of elevated magnesium concentrations on human coronary artery endothelial and smooth muscle cell (HCAEC, HCASMC) growth and gene expression. In the course of 24 h after incubation with magnesium chloride solutions (1 or 10 mM) intracellular magnesium level in HCASMC raised from 0.55 6 0.25 mM (1 mM) to 1.38 6 0.95 mM (10 mM), while no increase was detected in HCAEC. Accordingly, a DNA microarray-based study identied 69 magnesium regulated transcripts in HCAEC, but 2172 magnesium regulated transcripts in HCASMC. Notably, a signicant regulation of various growth factors and extracellular matrix components was observed. In contrast, viability and proliferation of HCAEC were increased at concentrations of up to 25 mM magnesium chloride, while in HCASMC viability and proliferation appeared to be unaffected. Taken together, our data indicate that magnesium halts smooth muscle cell proliferation and stimulates endothelial cell proliferation, which might translate into a benecial effect in the setC ting of stent associated vascular injury. V 2011 Wiley Periodicals,
Inc. J Biomed Mater Res Part B: Appl Biomater 100B: 4150, 2012.

Key Words: magnesium, bioabsorbable metal stent, stent, in-stent restenosis, cell cycle

How to cite this article: Sternberg K, Gratz M, Koeck K, Mostertz J, Begunk R, Loebler M, Semmling B, Seidlitz A, Hildebrandt P, Homuth G, Grabow N, Tuemmler C, Weitschies W, Schmitz K-P, Kroemer H. 2012. Magnesium used in bioabsorbable stents controls smooth muscle cell proliferation and stimulates endothelial cells in vitro. J Biomed Mater Res Part B 2012:100B:4150.

INTRODUCTION

Stents used as tubular implants for the mechanical support of stenotic arterial vessels were introduced in the 1990s. However, the initial concept of transluminal implantation of stainless steel coil springs was already described by Dotter in preclinical experiments in 1969.1 Stent endoprostheses are either self-expanding or balloon-expandable and are usually fabricated either as a wire mesh or a slotted tube. Typical materials of permanent bare metal stents (BMS) are stainless steel and cobalt-chromium alloys for balloon-expandable stents, and nickel-titanium alloys for self-expanding stents.2 Although BMS implantation demonstrated superiority over balloon angioplasty alone, numerous studies revealed that within 612 months BMS implantations are associated

with the formation of in-stent restenosis in about 1520% of all cases.3 This restenotic process is induced by vascular injury during stent implantation, leading to neointimal hyperplasia caused by proliferation and migration of smooth muscle cells.4,5 In rst attempts to decrease the incidence of in-stent restenosis the designs of BMS were modied and passive antithrombotic stent coatings were developed. However, a marked reduction of in-stent restenosis was only achieved with the introduction of drug-eluting stents (DES). DES effectively inhibit in-stent restenosis by controlled release of antiproliferative drugs, such as sirolimus (CypherV stent) and paclitaxel (TaxusV stent), directly to the site of vascular injury.6 These rst generation DES are very effective in preventing in-stent restenosis. However, it is
R R

*Both authors contributed equally to this work. Correspondence to: K. Sternberg (katrin.sternberg@uni-rostock.de) Contract grant sponsors: Landesregierung Mecklenburg-Vorpommern, Bundesministerium fur Bildung und Forschung; contract grant number: 03ZIK012

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being discussed that their use is associated with an increase in adverse clinical events, such as myocardial infarction. In this context, late thrombosis and delayed healing were identied as potential risks associated with the use of DES.79 Furthermore, cases of local hypersensitivity and inammation were reported.911 A promising approach to overcome the limitations of rst generation DES is the application of fully bioabsorbable stents, which could be completely replaced by tissue, and may even allow for a positive vascular remodeling.12 Apart from biodegradable polymers, such as polylactides,1318 stents based on the corrodible metals iron19 and magnesium20 have been proposed. So far, the magnesium stent is the only bioabsorbable metal stent investigated clinically as a coronary stent (PROGRESS-AMS trial).21 This clinical trial with a cohort of 63 patients demonstrated safe stent application and a safe absorption process after 4 months.22,23 Despite the initial clinical success of the magnesium stent, biocompatibility and biological action of the degradation products are still subject of scientic investigation.24 In this context, it is the purpose of the present in vitro study to assess the biocompatibility and pharmacological effects of the main alloy component magnesium (>90% w/w) with regard to cell proliferation and gene regulation of human coronary artery smooth muscle cells and endothelial cells.

the MTS reagent was adjusted to 333 lg/mL and the PMS reagent to 2.5 mM. Cell cultivation HCASMC and HCAEC were seeded at a density of 1 104 cells/mL in 96-well plates (200 lL per well) and cultivated in their respective growth media for 72 h. Then HCASMC and HCAEC were arrested by incubating the cells in basal medium with 0.1% (for HCASMC) or 0.5% (for HCAEC) FCS for 48 h. Subsequently the medium was removed and the different MgCl2 concentrations (1.5625, 3.125, 6.25, 12.5, 25, 50, 100 mM) were added to the basal medium with 5% FCS (for HCASMC and HCAEC). The cell arrest was released by stimulation with 25 ng/mL platelet-derived growth factor (PDGF, Sigma-Aldrich, Taufkirchen, Germany) or 25 ng/mL epidermal growth factor (EGF, Sigma-Aldrich), respectively. MTS cell viability assay At the end of the 24-h incubation time, the MgCl2 solutions were removed and 100 lL of the MTS medium were added to each well. The microtiter plate was incubated for 2 h at 37 C and 5% CO2. The yellow color of the tetrazolium salt (MTS reagent) changed towards a violet brown color of the formazan salt and was quantied with an ELISA reader (Anthos III, Anthos-Microsystems, Krefeld, Germany) at a wavelength of 492 nm and a reference wavelength of 690 nm. Cells kept in basal medium served as a control for restimulation of cell growth. BrdU cell proliferation assay At the end of the 24-h incubation time BrdU (10 lM) was added to each well and incubated for additional 18 h at 37 C and 5% CO2. BrdU incorporation was measured according to the supplier instructions. Cells not subjected to additional magnesium served as a reference, and cells kept in basal medium only served as a control for restimulation of cell growth. Determination of the intracellular magnesium concentration For measurement of intracellular magnesium concentrations cells were cultured in their respective growth media (PromoCell) and challenged with different MgCl2 solutions (1, 4, 10 mM) for 24 h. Afterwards, the cells were rinsed, detached, and resuspended in sodium-HEPES incubation buffer (135 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 20 mM HEPES, pH 7.4). Cells were loaded with Mag-Fura-2-AM (1 lM; Invitrogen, Carlsbad, CA) for 60 min at 37 C. Afterwards the labeled cells were incubated for 30 min in fresh sodium-HEPES incubation buffer at 37 C to allow complete de-esterication of intracellular AM esters. For the measurement 500,000 cells were washed and resuspended in 2 mL fresh sodium-HEPES incubation buffer and transferred to a 2-mL cuvette in a PerkinElmer LS-55 uorimeter. The cells were excited, alternately, at 340 and 380 nm and the emission intensity was recorded at 510 nm. Intracellular calibration of the Mag-Fura-2
IN VITRO MAGNESIUM EFFECTS ON SMOOTH MUSCLE AND ENDOTHELIAL CELLS

MATERIALS AND METHODS

Materials Magnesium was purchased as magnesium chloride (MgCl2) from Sigma-Aldrich (Taufkirchen, Germany). MgCl2 was dissolved in water to yield a concentration of 5 mol/L (M). The bioabsorbable magnesium stents were supplied by Biotronik AG (Bulach, Switzerland). Primary human coronary artery smooth muscle cells (HCASMC) and primary human coronary artery endothelial cells (HCAEC) were obtained from PromoCell GmbH (Heidelberg, Germany). Smooth muscle cell growth medium 2 containing 1 mM MgCl2, 5% fetal calf serum (FCS), 2 ng/mL recombinant human basic broblast growth factor, 0.5 ng/ mL recombinant human epidermal growth factor, 5 lg/mL recombinant human insulin, 620 pg/mL phenol red as well as antibiotics 50 ng/mL amphotericin B and 50 lg/mL gentamycin (pH 7.4) (with supplement mix) was supplied by PromoCell. For HCAEC magnesium-free endothelial cell growth medium MV (PromoCell) containing 5% fetal calf serum (FCS), 0.4% endothelial cell growth supplement/heparin, 10 ng/mL recombinant human epidermal growth factor, 1 lg/mL hydrocortisone, 620 pg/mL phenol red as well as antibiotics 50 ng/mL amphotericin B and 50 lg/mL gentamycin (pH 7.4) was adjusted to 1 mM MgCl2. For the MTS assays 100 mL Dulbeccos modied Eagle medium (DMEM, Applichem, Darmstadt, Germany) without phenol red was prepared, since phenol red in conjunction with the formazan salt mimics false positive viability results. The medium was supplemented with 10% FCS, 25 mM HEPES, 44 mM NaHCO3, and 100 U/mL penicillin/ streptomycin (pH 7.4). By adding 20 lL MTS/PMS reagent

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signals was done with the addition of digitonin (nal concentration 70 lM) to measure the maximal uorescence of the probe by releasing the indicator into the surrounding medium, followed by the addition of EDTA at a nal concentration of 10 mM to determine the uorescence, Fmin, in the absence of Mg2. The Mg2 concentration was calculated with the following equation25: Mg2 KD Q R Rmin Rmax R

using 1.5 mM as KD for the Mg2/Mag-Fura-2-complex. Q is the ratio of uorescence intensity of free Mag-Fura-2 to Mg bound Mag-Fura-2 at 380 nm. Ex vivo stent implantation and determination of the magnesium content in blood vessel tissue The magnesium uptake into the blood vessel wall after magnesium stent implantation was evaluated for n 6 magnesium stent systems (nominal dimensions 3.0 12 mm2, provided by Biotronik AG, Bulach, Switzerland) with a weight of $4.5 mg (magnesium content $90% 4.05 mg). Freshly harvested carotid artery sections from porcine cadavers obtained from the Leibniz Institute for Farm Animal Biology (Dummerstorf, Germany) were used as model vessels. Ex vivo, each stent was deployed into a 25 mm porcine artery section by balloon-expansion (8 bar, 15 s) to an inner diameter of 3.0 mm. The stent-implanted artery sections were placed in 5 mL incubation media (DMEM containing 10% fetal bovine serum and 1% penicillin/streptomycin) at 37 C. In compliance with the MgCl2 incubation time in the cell culture studies the magnesium accumulation was analyzed after 24 and 48 h. For control purposes sections of the same arteries without stents were incubated under identical conditions. After the intended incubation periods the arteries were removed from the media and thoroughly rinsed. Incubated arteries as well as frozen samples were dried at 60 C for 24 h and dry mass was determined. Afterwards, the sections were dissolved in 65% (w/ w) nitric acid (HNO3) under heating. Magnesium content of samples of the dissolved arteries and incubation media was determined by atomic absorption spectroscopy (AAS, atomic absorption spectrophotometer PU 9200, Philips GmbH, Hamburg, Germany) against magnesium ICP standard 1000 mg/L CertiPUR (VWR International, Darmstadt, Germany). All samples and standards contained 4% (v/v) HNO3 in their nal dilution. Samples were atomized in an air/ acetylene ame (fuel ow 1.1 L/min) and atomic absorption was determined at a wavelength of 285.2 nm (slit width 0.5 nm). Preparation of RNA (transcriptome prole and real time RT-PCR) HCAEC and HCASMC were seeded into 6-well plates and cultured for 48 h in their respective culture media containing 1 mM MgCl2, followed by incubation for 24 h in culture media containing either 1 mM (control) or 10 mM MgCl2.

Total RNA was isolated performing a modied phenol extraction using the TRIzol reagent (Invitrogen)26 followed by total RNA purication by RNeasy mini kit (Qiagen, Hilden, Germany). RNA concentration was measured using the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies) and RNA purity and quality was assessed by gel electrophoretic separation using the lab-on-chip capillary electrophoresis technology (Bioanalyzer 2100, Agilent Technologies, Waldbronn, Germany). Only RNA samples with a RNA Integrity Number (RIN)27 greater than 9.5, 260/280 nm ! 1.8 were used for microarray analysis and real-time RT-PCR. Target preparation and array processing (transcriptome prole) To elucidate genes whose expressions are signicantly changed at the level of mRNA and which may contribute to the molecular mechanisms underlying the physiological changes mediated by excess of magnesium, global mRNA proling using GeneChip Human Genome 133 Plus 2.0 Arrays (Affymetrix, Inc., USA) was carried out. For each condition, global gene expression of two replicates was interrogated, respectively. Target preparation and target hybridization were performed according to the manufacturer instructions. In brief, 5 lg of target RNA was reverse transcribed into cDNA and consecutively transcribed into biotinylated cRNA using the one-cycle target labeling and control reagents kit (Affymetrix). cRNA concentration and purity was checked using the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies) and cRNA quality was assessed by gel electrophoretic separation using the Bioanalyzer 2100 (Agilent). Hybridization was carried out at 45 C for 16 h. Staining and scanning was performed by streptavidin phycoerythrin using the Fluidics Station 450 (Affymetrix) and Gene Chip Scanner (Affymetrix). For calculation of probe specic pixel intensities, Affymetrix CEL les were generated from uorescence intensities using the GCOS 5.0 software package (Affymetrix). Quality control of all arrays was performed by inspecting the corresponding scan images and by carefully reviewing external and endogenous controls. For all processed arrays, the available control parameters matched the default threshold tests and were considered to be of adequate quality. Microarray expression analysis Calculation of expression summaries and ltering of differentially expressed genes was done using the Gene Spring GX 7.3 expression analysis system for gene expression data analysis (Agilent Technologies, Santa Clara, CA). After importing CEL-les, probe set specic signals were summarized using the robust multiarray average with GC-content background correction (GC-RMA) algorithm. Normalization was carried out by GC-RMA preprocessor integrated quantile normalization thereby generating normalized signal intensities for each probe set. To generate fold changes, ratios were calculated by dividing the mean normalized signal intensity of 10 mM MgCl2 by the mean normalized signal of the baseline (1.0 mM MgCl2). A change !2.0-fold

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FIGURE 1. Cell viability and proliferation of HCAEC (*) and HCASMC (n) in response to MgCl2 after 24 h incubation. Viability was measured by MTS assay with the 1 mM group as the 100% reference point (a). Cell proliferation was measured by the BrdU assay with the 1 mM group as the 100% reference point (b). Values are given as mean 6 SD of at least four independent experiments with three replicates each. Differences between HCAEC and HCASMC at a given MgCl2 concentration were analyzed using the two-tailed Students t-test. p < 0.05 was considered signicant (*).

was used as cutoff for further analyses including Ingenuity Pathway Analysis software (IPA, Ingenuity Systems, Redwood City, CA).

at 37 C for 90 min and measured at a wavelength of 405 nm.

Cell cycle analyses and apoptosis assay For cell cycle analyses cells were seeded as described above and treated with different MgCl2 concentrations. The cells were harvested and analyzed for DNA content by ow cytometry (FACScan cytometer, CellQuest software; Becton Dickinson, Heidelberg, Germany) as described previously.28 The percentage of cells in different phases of the cell cycle was determined using the CellQuest software. For determination of apoptosis a caspase 3 activity colorimetric assay (R&D Systems GmbH, Wiesbaden-Nordenstadt, Germany) was used. Controls and MgCl2 treated HCASMC were harvested and lysed in lysisbuffer (50 mM Tris-HCl, 100 mM NaCl, 0,1% TritonX-100, 5 mM EDTA, pH 7.4) for 10 min on ice. Afterwards lysates were centrifuged at 10,000g for 1 min and protein content of the supernatant determined using the BCA protein assay (Thermo Fisher Scientic Inc., Rockford, IL). Totally, 100 lg proteins were loaded onto a 96-well microplate and incubated with 2 reaction buffer including DTT. After addition of caspase 3 colorimetric substrate (DEVD-pNA) the plate was incubated

Real time RT-PCR Cyclin D2 (Hs00277041_m1), Cyclin E2 (Hs0151894_m1), CDK2 (Hs00608082), as well as p21 (Hs00355782_m1) mRNA levels were determined by TaqMan quantitative real time PCR applying the ABI Prism 7900 sequence detector system (Applied Biosystems, Foster City, CA). The isolated RNA was reversely transcribed using random nonamer primers and the Eurogentec reverse transcription kit (San Diego, CA). The cDNA (5 ng/lL reversely transcribed RNA) was amplied using a PCR mastermix containing 45 mM Tris-HCl (pH 8.4), 115 mM KCl, 7 mM MgCl2, 460 lM dNTPs, 9% glycerol, 2.3% ROX reference dye (Invitrogen, Carlsbad, CA) and 0.035 U/mL Platinum Taq DNA polymerase (Invitrogen). The relative quantication of each target gene was analyzed using the 2-DDCT method in which 18S rRNA levels (18S rRNA endogenous control; Applied Biosystems) served as the endogenous reference, where DCT is the difference in the CT values of gene of interest and the endogenous reference and DDCT is the mean DCT of the samplemean DCT of the control sample (used as calibrator).

FIGURE 2. Intracellular magnesium concentrations of HCASMC (a) and HCAEC (b) incubated in the presence of MgCl2 (1, 4, and 10 mM) for 24 h were measured by a uorometric Mag-Fura 2-based assay. Values are given as mean (SD for at least n 8 of three independent experiments. Statistical analysis was performed using a one-way ANOVA followed by Dunnetts post hoc test (against 1 mM MgCl2). p < 0.05 was considered signicant (*).

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TABLE I. Magnesium Concentrations [lmol/g artery] After Ex Vivo Stent Implantation into Porcine Blood Vessels Incubation Time 24 h 48 h Control 7.0 6 1.2 8.2 6 1.2 Stent 15.6 6 2.0 17.3 6 2.5 Fold Change 2.2 2.1

the medium was observed to be 0.55 6 0.25 mM. Elevation of extracellular magnesium to 10 mM was followed by a 2.5-fold increase in intracellular magnesium concentration to 1.38 6 0.95 mM (p < 0.05). In contrast, no increase in intracellular magnesium concentration was observed for HCAEC (Figure 2). Magnesium accumulation in the blood vessel wall To obtain insights into the putative changes of the magnesium concentration in the blood vessel wall due to a magnesium stent implantation porcine vessel sections provided with magnesium stents were cultured ex vivo under static conditions at 37 C. Atomic absorption spectroscopy revealed a twofold increase in tissue magnesium concentrations after 24 and 48 h (Table I). Gene expression analyses In order to obtain information about gene expression changes based on the results of the magnesium accumulation experiments and the marked differences in cell proliferation and viability between HCAEC and HCASMC at 110 mM MgCl2, expression analysis was performed using 10 mM in relation to 1 mM MgCl2 as a reference. The combination of GC-RMA based signal extraction and a cut-off change of !2.0-fold resulted in the identication of 69 transcript-specic probe sets in endothelial cells and 2172 transcript-specic probe sets in smooth muscle cells after incubation with 10 mM MgCl2 for 24 h. Our results indicate that global gene expression appears almost unchanged in HCAEC, whereas addition of 10 mM MgCl2 caused a massive change of gene expression in HCASMC, suggesting a comprehensive reprogramming of smooth muscle cell physiology in the presence of 10 mM MgCl2 (Figure 3). With the purpose to obtain information about functional processes and pathways affected by gene expression changes in HCASMC, the identiers of all differentially

The vessels with implanted magnesium stents were incubated for 24 and 48 h in a static chamber (each with n 6). Control tissue of the same vessel was kept under identical conditions (n 6).

Statistical analyses Data were analyzed using GraphPad Prism 5.01 software (GraphPad Software, San Diego, CA). To evaluate statistical signicance the two-tailed Students t-test (comparison of two groups) or one-way ANOVA followed by Dunnetts post hoc test (comparison of more than two groups against the control) were used. Values were considered signicant at p < 0.05.
RESULTS

Impact of magnesium on cell viability and proliferation The results of the cell viability assays (MTS) using HCAEC or HCASMC demonstrate that for a range of magnesium concentrations endothelial cells benet from higher levels of magnesium, while viability of smooth muscle cells was unchanged compared with the control [Figure 1(a); maximum effect of 115% at 12.5 mM MgCl2]. BrdU assays demonstrated a similar benecial effect of magnesium on HCAEC proliferation for concentrations ranging from 6.25 to 25 mM MgCl2 [Figure 1(b); maximum effect 145% at 12.5 mM MgCl2]. Intracellular magnesium concentration To determine the changes of intracellular magnesium in dependence of elevated extracellular concentrations, HCAEC as well as HCASMC were incubated with different MgCl2 concentrations for 24 h. In HCASMC the mean intracellular magnesium concentration in the presence of 1 mM MgCl2 in

FIGURE 3. RNA from two sample replicates of HCASMC (a) and HCAEC (b) was analyzed by Affymetrix Genechip HG U133 plus 2.0 arrays. The scatterplots show a distinct change of gene expression in HCASMC, but only minor changes in HCAEC after growth in the presence of 10 mM MgCl2 (y-axis) compared with growth with 1 mM MgCl2 (x-axis). Gene-specic probe sets with expression changes varying more than twofold are indicated in red (increased) or green (decreased). [Color gure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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TABLE III. Expression Fold Changes of Growth Factor and Growth Factor Receptor Genes in HCASMC (10 mM MgCl2, with 1 mM MgCl2 Serving as Reference) Gene VEGFA TGFB2 FGF5 PDGFC FGF1 ANGPT2 HGF PDGFD FLT1 EGFR TGFBR2 IGF2R FGFR1 Expression (Fold Change) 5.7 3.4 2.9 2.6 2.1 2.2 2.5 2.8 2.0 3.8 7.6 2.0 3.4 Description Vascular endothelial growth factor Transforming growth factor B2 Fibroblast growth factor 5 Platelet-derived growth factor C Fibroblast growth factor 1 Angiopoetin 2 Hepatocyte growth factor Platelet-derived growth factor D VEGF receptor Endothelial growth factor receptor Transforming growth factor receptor R1 Insulin-like growth factor receptor FGF receptor

FIGURE 4. The 10 most probable biological functions of HCASMC identied by IPA via the ratio of regulated vs. unregulated transcripts within the overall set of transcripts, and allotted to the respective functions probably involved in the reaction to an increase in magnesium concentration.

expressed genes were uploaded into IPA and were mapped to their corresponding gene objects in the IPA knowledge database. Specic networks and biological functions were calculated based on their connectivity and functionality. From the list of 2172 probe sets, 1102 genes could be localized to functions, pathways or lists and 1274 genes were eligible for network generation. Based on statistical testing implemented in the IPA software, the ten biological functions most affected by magnesium accumulated in HCASMC were identied (Figure 4). The ve most affected functions are: (i) cancer, (ii) cell death, (iii) cell cycle, (iv) cellular growth and proliferation, and (v) organismal survival. Furthermore, from 84 identied networks with scores in the range of 142 for HCASMC incubated with 10 mM MgCl2 compared with control conditions, the two most signicant were associated to gene expression, cell signaling, cell cycle and cellular movement, connective tissue development and function, cell death (Table II). Detailed inspection of the microarray experiments revealed that many genes associated with growth factors, their receptors as well as extracellular matrix components are regulated in HCASMC after incubation with 10 mM MgCl2. Table III lists changes of growth factors and growth factor receptors. Most

prominently VEGF-A expression is reduced 5.7-fold. Furthermore, TGFb2, FGF1, and FGF5 are down regulated as well. PDGF isoform C could be demonstrated to be down regulated (2.6-fold) whereas the expression of isoform D was up regulated by a factor of 2.8. HGF and Ang2 (ANGPT2) expression was increased. No change in expression was seen for FGF 2 to 4 and 10 to 23, EGF, VEGF-C, and PDGFBB. For growth factor receptors, the transforming growth factor beta receptor 2 expression was found decreased by a factor of 7.6, VEGF receptor expression (FLT1) by 2.0, EGF receptor by 3.8, IGF 2 receptor by 2.0, and FGF receptor by 3.4, while no regulation was observed for angiotension II receptors 1 and 2, PDGF receptor alpha and the VEGF receptor 3 (FLT4). Regarding changes in extracellular matrix components and regulators, Table IV shows the impact of MgCl2 on the expression of various collagen subtypes, most notably collagen type I (8.7-fold). With the exception of collagen types III and XIV, which are up regulated by 2.0 and 3.4, respectively; all other collagens were observed to be down regulated by factors ranging from 2.0 to 8.7. In addition,

TABLE II. The 10 Most Signicant Networks and Associated Network Functions in HCASMC Top Functions of Networks Gene expression, cell signaling, cell cycle Cellular movement, connective tissue development and function, cell death Behavior, cancer, cell cycle Cell cycle, amino acid metabolism, post-translational modication Cell cycle, cellular movement, amino acid metabolism Cell cycle, embryonic development, hair and skin Development and function Immune and lymphatic system development and function, cellular growth and proliferation, Hematological system development and function Cellular assembly and organization, amino acid metabolism, molecular transport Cell death, dermatological diseases and conditions, cancer Organismal injury and abnormalities, cell morphology, nervous system development and function Score 42 42 42 40 40 38 38 38 33 32 Focus Molecules 34 34 34 33 33 32 32 31 30 33

Networks of genes were algorithmically generated with the IPA software based on their connectivity and assigned a score, which refers to the statistical signicance (negative logarithm of the p value). Focus molecules refer to the number of genes in each network.

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TABLE IV. Expression Fold Changes of Collagens in HCASMC (10 mM MgCl2, with 1 mM MgCl2 Serving as Reference) Gene COL1A1 COL4A1 COL4A2 COL5A1 COL6A1 COL12A1 COL15A1 COL22A1 COL3A1 COL14A1 Expression (fold change) 8.7 2.7 3.3 2.0 3.7 2.0 2.4 3.7 2.0 3.4

magnesium did not result in signicant changes of cell cycle phases. However, we did observe a slight increase in apoptosis of HCASMC with increasing magnesium concentrations to 1.26 6 0.15 and 1.55 6 0.38-fold (4 and 10 mM, respectively) compared to 1 mM MgCl2, which reached statistical signicance for 10 mM [Figure 5(D)]. For doxorubicin, which was used as a positive control, a 3.1 6 0.24-fold increase in apoptosis was observed.
DISCUSSION

our analysis revealed a number of integrin subunits which appear to be differentially expressed, with subtypes A4, A11, and B1 being down regulated by factors of 2.1, 3.0, and 2.3, respectively. In contrast, up regulation was observed for subunits A10 (2.4) and B8 (2.6). Expression of bronectin (FN1) as well as expression of the matrix metalloproteinases 3, 10, 12, and 14, all involved in wound repair and cell migration, was also substantially down regulated (3.3 to 8.4). Taken together, these ndings suggest a comprehensive reprogramming of HCASMC in the presence of 10 mM MgCl2 most likely towards decreased abilities of proliferation, cell growth and cellular movement.

Cell cycle and apoptosis analyses Within the group of highly regulated networks and biological functions (Figure 4) cell cycle genes are regulated. Therefore we performed more detailed real time RT-PCR based analyses of regulated genes involved in cell cycle control. These analyses veried the down regulation of the cyclin E2 (between 17 and 21%) and CDK2 (between 57 and 73%), both necessary for progression from G1- to Sphase [Figure 5(B), diagram] for 10 and 4 mM MgCl2, respectively. P21 mRNA is up regulated (between 261 and 305%) and exerts an inhibitory function on cyclin E2 further enhancing the retarding effect on the cell cycle. In contrast, cyclin D2 mRNA is up regulated (between 276 and 304%) by 10 and 4 mM MgCl2, respectively, and should have a stimulating effect on HCASMC division [Figure 5(B), diagram]. There is no statistical signicant difference between 4 and 10 mM MgCl2 in up or down regulating cyclin D2 and p21 or cyclin E2 and CDK2, respectively. The data from real time RT-PCR experiments [Figure 5(B), diagram] conrm the data from microarray hybridization experiments [Figure 5(B), table]. Taken together there is no effect on cell cycle progression [Figure 5(C)]. None of these changes in expression was seen in HCAEC. In order to evaluate the effects of magnesium on the cell cycle itself, the relative percentages of HCASMC in each phase were quantied by propidium iodide staining and subsequent FACS analysis. The relative percentages of smooth muscle cells in sub G0/G1, G0/G1, S, and G2/M at various magnesium concentrations are illustrated in Figure 5(C). Treatment with

Magnesium, the most abundant intracellular divalent cation, is involved in fundamental biological functions. Supplementation of magnesium can convey cardiovascular protective effects just as reduction of carotid intimal thickening29 and improvement of endothelial function.30 Based on this, this article investigated any direct biological effects of the magnesium released from biodegradable magnesium alloy stents on gene expression, viability, and proliferation of coronary endothelial (HCAEC) and smooth muscle cells (HCASMC). Results of the presented in vitro experiments demonstrate that magnesium concentrations ranging from 6.25 to 25 mM MgCl2 enhance HCAEC proliferation (Figure 1). Furthermore, the gene expression analyses show distinct changes in mRNA abundance in HCASMC, but only minor changes in HCAEC, after incubation with 10 mM MgCl2 (Figure 3). In this context, it can be stated that magnesium affects HCASMC gene expression in a way that indicates interference with major elements of neointima formation, e.g., down regulation of growth factor receptors and of components of the extracellular matrix (Tables III and IV). For the gene expression studies the extracellular magnesium concentration of 10 mM was chosen according to the following considerations: a minimum of about 1 mM, a maximum of about 200 mM, and a practical ex vivo result giving a twofold increase in tissue concentration following implantation of a magnesium stent. The cited minimum is based on the concentration of magnesium in human serum (0.51.5 mM31), the theoretical maximum is determined by total amount of magnesium incorporated into such bioabsorbable stent ($4 mg, resulting in a theoretical maximum of 197 mM local concentration [tissue volume 0.83 mL (length: 1.25 mm, inner diameter: 3.0 mm, outer diameter: 5.5 mm)]), and the ex vivo result came from spot sample measurements in porcine vascular tissue in presence or absence of a magnesium stent, which indicated a twofold increase in magnesium tissue concentration (Table I). Further validity to the use of an extracellular magnesium concentration of 10 mM was given by the observations that, in accordance with results from the ex vivo stent implantation experiment, an incubation of HCASMC with 10 mM MgCl2 resulted in an about 2.5-fold increase of intracellular magnesium as compared with incubation at 1 mM MgCl2 (Figure 2). Interestingly, our in vitro experiments on HCAEC and HCASMC proliferation and viability demonstrate that magnesium differently affects these cell types in a concentration range of 6.2525 mM MgCl2 (Figure 1). Under these conditions proliferation and viability of HCASMC is lower than

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FIGURE 5. Inuence of MgCl2 on cell cycle regulation. Effect of 10 mM MgCl2 on genes involved in G1/S phase cell cycle regulation based on IPA. Down regulated genes are presented in green, up regulated genes in red (A). Quantication of mRNA abundance of genes involved in cell cycle regulation using real-time PCR (B, diagram) and microarray hybridization data for these genes induced by 10 mM MgCl2 (B, table). Effect of MgCl2 on cultured HCASMC cell cycle phases after a 24-h exposure to different MgCl2 concentrations. Data are from ow cytometric analyses of cultured HCASMC labeled with propidium iodide. Data are presented as percentages of cells in sub-G0/G1, G0/G1, S, and G2/M phase of three independent experiments (C). Caspase 3 activity of HCASMC exposed to different MgCl2 concentrations as well as doxorubicin (1 lM Dox) for 24 h (D). Data are presented as mean (SD for n 4. Statistical analysis was performed using a one-way ANOVA followed by Dunnetts post hoc test (against 1 mM MgCl2). p < 0.05 was considered signicant (*). [Color gure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

proliferation and viability of HCAEC irrespective of an initial cell cycle arrest by serum deprivation (data not shown). This might in part be explained by the lack of increased intracellular magnesium ions in endothelial cells after incubation of endothelial cells at 10 mM MgCl2, most likely reecting the tight regulation of magnesium levels of endothelial cells via regulation of magnesium inux, efux, intracellular compartmentalization, or a combination thereof.32 Accordingly, whole genome gene expression analyses revealed 69 differentially expressed transcripts for HCAEC, but 2416 regulated HCASMC transcripts after 24 h of incubation with 10 mM MgCl2 compared with control conditions. Obviously, stimulation of HCAEC proliferation by

MgCl2 [Figure 1(b)] is not associated with substantial changes in mRNA abundance [Figure 3(b)], but is most likely due to a regulatory physiological function of magnesium, e.g., cofactor role of magnesium for numerous enzymatic reactions.33,34 Furthermore, magnesium is involved in regulation of ion transport processes by carrier proteins and channels and thereby modulates signal transduction processes.35,36 For differentially expressed genes in the smooth muscle cells, the automated pathway analysis identies functions including cancer, cell death, cell cycle, cellular growth and proliferation, and organismal survival as most likely being affected by MgCl2 (Figure 4). These functions cover
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genes involved in processes attributed to cell growth, cell division, migration, and regulation of extracellular matrix all part of the current understanding behind neointimal hyperplasia. In more detail, we monitored the down regulation of several growth factor receptors and associated growth factors along with reduced transcript levels of metalloproteinases, bronectin and at least seven different kinds of collagen as a direct adjustment on an increased extracellular magnesium concentration in smooth muscle cells (Tables III and IV). Considering the major contribution of matrix metalloproteinases to cell migration37 and their extensive down regulation, magnesium might lead to a net reduction of smooth muscle cell migration. Although numerous cell cycle regulatory genes and cell proliferation associated genes in smooth muscle cells were found down regulated by gene expression analyses [Figure 5(A)] these observations did translate into halting smooth muscle cell proliferation [Figure 5(C)].
CONCLUSION

Magnesium at a concentration of 10 mM regulates HCASMC gene expression in such a way that migration and proliferation are down regulated resulting in a quiescent appearance of HCASMC. In contrast, proliferation of HCAEC is stimulated by 10 mM magnesium with minor effects on gene expression. Taken together, the differential effect of magnesium on HCASMC and HCAEC might well explain the good in vivo performance of the bioabsorbable magnesium stent.2123 The observed stimulation of HCAEC proliferation in a concentration range of 6.2525 mM magnesium emphasizes the demand for controlled magnesium stent corrosion and the related magnesium ion release. Potential approaches in this direction may be magnesium stent coatings based on corrosion inhibiting ceramics or hydrophobic polymers, as recently suggested by Lu et al.38,39
ACKNOWLEDGMENTS

The authors thank Martina Nerger and Babette Hummel for their expert technical assistance. The authors are furthermore grateful to Prof. Gerhard Hennighausen and Dr. Claus Harder for helpful notes and suggestions. Additionally, thanks are given to the Biotronik AG for the provision of the bioabsorbable magnesium stents.
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