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SUPPLEMENTARY FILE
Bis(aspirinato)zinc(II) complex successfully inhibits carotid arterial neointima formation after ballon-injury in rats
Péter Hegedűs MD1,2, Sevil Korkmaz PhD1, Tamás Radovits MD, PhD2, Harald Schmidt1, Shiliang Li MD1, Yutaka Yoshikawa PhD3; Hiroyuki Yasui PhD3, Béla Merkely MD, PhD2, Matthias Karck MD, PhD1, Gábor Szabó MD, PhD1
1Department of Cardiac Surgery, University of Heidelberg, Germany
2Heart and Vascular Center, Semmelweis University, Hungary
3Department of Analytical and Bioinorganic Chemistry, KyotoPharmaceuticalUniversity, Kyoto, Japan
Corresponding author: Péter Hegedűs MD
Laboratory of Cardiac Surgery
Department of Cardiac Surgery
University of Heidelberg
INF 326, 69120 Heidelberg, Germany
Tel.: +49-6221-566246 Fax: +49-6221-566246
Email:
Animals
For the experiments young male Sprague-Dawley rats were used with the body weight of 300 to 330 g. Animals were obtained from Charles River, Sulzfeld, Germany.They were housed in a room at a constant temperature of 20±2°C with 12-hour light/dark cycles and were fed a standard dry laboratory rat diet and water ad libitum.
All procedures concerning animals were conformed to the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86–23, revised 1996) and the German animal protection code. Approval was also granted by the local ethics review board (G-135/13).
Experimental groups
Experimental animals were randomly assigned into 4 experimental groups (12 animals per group). Right carotid arteries of the animals in the treated groups underwent to balloon-injury and 18 days postoperative medication with ZnCl2, ASA, Zn(ASA) 2 or vehicle, respectively. Left carotid arteries of the vehicle group underwent no operation and were analysed asabsolute controls(See Online table 1).After 18 days animals were harvested and two third of the carotids from each animalwere saved in 4% paraformaldehide solution for histology, the other third wasfrozen and stored in -80 oC for PCR analysis.
Drugs and treatment
Zinc complex of aspirinate
Zn(ASA)2 was provided by the research group of Prof. Hiroyuki Yasui, Department of Analytical and Bioinorganic Chemistry, KyotoPharmaceuticalUniversity, Kyoto, Japan. The molecular formula is C18H14O8Zn2, named "zinc bis[2-(acetyloxy)benzoate" with the molecular weight of 423.708 g/mol.The complex was diluted in Polyethylene-Glycol to the final concentration of 105mg/ml. This solution contained 15 mg Zinc and 90 mg acetylsalicylate. It was administrated by oral gavage, in the daily dosage of 105 mg/kg Zn(ASA)2 (=15 mg/kgBW Zn and 90 mg/kg ASA) for 18 days.
Zinc-chloride
ZnCl2 (Sigma-Aldrich, Seelze, Germany) was diluted from powder form in saline (high water solubility) so that the final concentration was 31 mg/ml ZnCl2. This solution contained 15 mg Zinc and 16mg Cloride. It was administrated by oral gavage, in the dosage of 31mg/kgBW ZnCl2 (=15 mg/kgBW Zn) daily for 18 days.
Acetylsalicylate
ASA (Sigma-Aldric, Seelze, Germany) was diluted from powder form in physiologic saline so that the final concentration was 90 mg/ml ASA. It was daily administrated by oral gavage, in the dosage of 90 mg/kgBW ASA for 18 days.
Polyethylene-Glycole (Macrogol 400)
PEG 400 was obtained from Caesar & Loretz, Hilden, Germany was used as diluent of ASA and ZnCl2. As placebo treatment of the vehicle group it was daily administrated by oral gavage for18 days.
Hematoxylin-Eosin Staining
The hematoxylin eosin staining is a well established method and belongs to the routine stainings. Nucleus and ribosome rich regions are stained by Hematoxylin to blue-vilolet, meanwhile intra and extracellular fibrillar compontents of the matrix become red by Eosin. HE-staining was used for the morphometric analysis of the vessels. Three stained cross-sections of each animal were representing the proximal, middle and distal regions of the operated vessels, and were analysed by using computer-aided planimetry (Q-Win; Leica, Solms, Germany). Luminal, intimal and medial dimensions were measured by using the internal and external elastic laminae as delimeters. The percentage of stenosis was calculated by using the ratio between the neointimal area and the original luminal area. In addition, the neointimal/medial ratio was calculated for each animal, and then again the mean was calculated for the different experimental groups and were compared.
Sirius Red / Fast Green staining for collagen detection
Sirius red staining was performed for each carotid artery (4x12 sections). Sections were subjected to xylene (3 x 5 min) for deparaffinization, hydrated in serial washes of decreasing ethanol concentrations (100%, 100%, 96%, 70% for 2 min each) and rinsed in distilled water. Staining was performed following the instructions of the manufacturer (Chondrex inc, Redmond, WA, US). Sections were incubated in dye solution for 30 min than rinsed with distilled water and dye extraction solution. Slides were mounted with Eukitt (Kindler GmbH, Görgeshausen, Germany).One representative cross-section per carotid artery was analyzed. Sections were examined using light microscopy (Research Microscope BX51, Olympus). Pictures were generated with a digital camera (ColorView I, Olympus) connected to the microscope and cell^A imaging software (Olympus). Brightness, saturation, contrast levels and RGB levels were set at the beginning of the study and kept constant throughout examination of all sections. Four pictures per carotid cross-section from the right, left, upper and lower segment were recorded at a magnification of x400. Image J imaging software (Version 1.43g, Open Source, was used to determine the collagen area fraction in the media of each segment by measuring the ratio of purple and red pixels to the total number of pixels in the media. The mean of the four segment values was calculated to provide a single value for each cross-section and thus for each carotid artery.
Immunohistochemistry
Immunohistochemistry was performed for on each carotid artery. The samples were reacted with alpha smooth muscle actin (anti-αSMA mouse monoclonal IgG, αSMA immunohistology kit, Sigma-Aldrich, Steinheim, Germany) and matrix metalloproteinase 9 (anti-MMP9 mouse monoclonal IgG, Life Span Biosciencesand) primary antibodies. The optimal dilutions were evaluated in a pilot study. For MMP-91:50antibody-dilution was used, and for αSMA prediluted antibody solution was provided in the kit. Negative controls were carried out by omitting the primary antibody.Sections were deparaffinized in xylene (3 x 5min), rehydrated in decreasing concentrations of ethanol (100%, 100%, 96%, 70% for 2 min each), rinsed in distilled water, placed in 10mM citrate buffer (pH 6.0) and cooked in the microwave for 20 min. Then they were placed at room temperature, allowed to cool for approximately 20 min, rinsed in distilled water and washed in TBS for 10 min. Afterwards sections were incubated with primary antibody in antibody diluted in PBS for 1 hour followed by another rinse in TBS (2 x 5 min). MMP-9 staining was completed using the labeled substrate avidin biotin technique (LSAB+ System-HRP from DAKO North America, Inc.Hamburg, Germany) and developed with brown chromogen substrate (DAB+). αSMA staining was performed as described in the protocol provided by the manufacturer. Target antigen was detected as red staining. Subsequently, all sections were rinsed in distilled water, counterstained with haematoxylin, rinsed again in distilled water and washed with tap water for at least 5 min. Finally, sections were dehydrated through a graded series of ethanol, cleared in xylene, mounted with Eukitt (Kindler GmbH, Görgeshausen, Germany) and examined by light microscopy (see above). One representative cross-section per carotid artery was evaluated for all immunohistological stainings.Four windows from the right, left, upper and lower segment of each cross-section were analyzed at a magnification of x 400 with ImageJ (Version 1.43g, Open Source, The 4 selected areas (windows) of the sections were assigned to area and intensity scores. Semiquantitative analysis was performed separately for the media and the neointima. An intensity score (0 = no positive staining, 1 = weak staining, 2 = intermediate staining, 3 = extensive staining) and an area score ( 1 = ≤ 10% positive cells, 2 = 11-50% positive cells, 3 = 51-80% positive cells, 4 = > 80% positive cells) were assigned to each window by an observer blinded to treatment and a total score was calculated (intensity score multiplied by area score, 0-12). In the rare cases, when inhomogenous intensity was observed within one window, an average intensity score was assigned.Finally the mean of the four windows was calculated to provide a single value for each specimen.
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)
TUNEL is a common method for detecting DNA fragmentation that may result from apoptosis or oxidative strass caused DNA damage by detecting double chain breakings through thier free 3'-OH endings. Sections were de-paraffinized with xylene and passaged through decreasing concentrations of ethanol, washed with distilled water and incubated with 50 µl of Terminal deoxynucleotidyl Transferase (TdT) enzyme and TUNEL Reaction mixture for 1 h at 37°C in the dark (Roche Diagnostics, Mannheim, Germany). The sections were then washed with PBS (1×) for 3×5 min. The slides were mounted using 4′, 6-diamidino-2-phenylindole (DAPI)-Fluoromount-G™ (SouthernBiotech, Birmingham, AL, USA), covered with cover glass and analyzed under a fluorescence microscope. The number of TUNEL-positive cells was expressed as the ratio of DAPI-TUNEL double-labeled nuclei to the total number of nuclei stained with DAPI. Cells were counted in four fields characterizing each specimen), and an average value was calculated for each experimental group. The evaluation was conducted by an investigator blinded to the experimental groups.Four labelled cross-sections of each animal were representing the proximal, mid-proximal, mid-distal and distal regions of the operated vessels. Cells with clear red labeling were counted in 1000 µm2 of neointimal tissue segments in all 3 sections and mean was calculated for each animal. Mean values were calculated again for each experimental groups and were compared among unoperated and treated groups.
Evaluation of qRT-PCR
Efficiency of the PCR reaction was confirmed with standard curve analysis. Every sample was quantified in duplicate, normalized to β-actin expression. Primers were obtained from TIB Molbiol (Berlin, Germany), their sequences and UPL probes used are represented on online Table II. Evaluation was performed with LightCycler 480 SW1.5 software (Roche, Mannheim, Germany).Efficiency of the PCR reaction was confirmed with standard curve analyis. Every sample was quantified in duplicate, normalized to β-actin expression. Expression of genes involved in inflammation, proliferation and extracellular matrix metabolism (A20, E-selectin, Il-6, iNOS, MCP-1, MMP-2, MMP-9, NF-κB, PCNA, TGF-β)was determined. Results form every groups were illustrated as normalized to the uninjured control values.
Online Table I
No / Group names / Carotid arteriotomy / Group size1 / Zn15 mg/kg per os / yes / 12
2 / ASA 90 mg/kg per os / yes / 12
3 / Zn(ASA)2 105 mg/kg per os / yes / 12
4 / Vehicle (PEG) / yes / 12
Online table I: Experimental groups of animals
Online Table II
The sequences for the forward (F) and reverse (R) primers (from 5’ to 3’) and Universal Probe Library (UPL) probes
Assay / Sequence / UPL probesα-actin / F: 5’-CGCCGATCCAGACAGAAT
R: 5’-CCCAGCACCATGAAGATCA / 25
β-actin / F: 5’-GCCTGGATGGCTACGTACA
R: 5’-CTAAGGCCAACCGTGAAAAG / 115
E-selectin / F: 5'-TCCTCTGGAGAGTGGAGTGC
R: 5′-TCAAGCTTTACATTCAACCACA / 68
Il-6 / F: 5'-ACAACATCAGTCCCAAGAAGG
R: 5′-CCCTTCAGGAACAGCTATGAA / 127
iNOS / F: 5'-CATGGTGAACACGTTCTTGG
R: 5′-CCCAGAGTCTCTAGACCTCAACA / 89
MCP-1 / F: 5'-AGCATCCACGTGCTGTCTC
R: 5′-TGGCAGTGCACAAACACC / 62
MMP-2 / F: 5’-AGCACCCTTGAAGAAATAGCTG
R: 5’-TGATAACCTGGATGCAGTCG / 77
MMP-9 / F: 5’-GGTCAGGTTTAGAGCCACGA
R: 5’-CCTCTGCATGAAGACGACATAA / 42
NF-κB / F: 5′-CCGGGATGGCTTCTATGAG
R: 5′-CACTGGATCCCCAGGTTCT / 113
PCNA / F: 5’-TGAACTTTTTCACAAAAGCCACT
R: 5’-TGTCCCATGTCAGCAATTTTA / 94
TGF-β / F: 5’-ACGCCAGGAATTGTTGCTAT
R: 5’-TCAGACATTCGGGAAGCAGT / 56
TNFAIP3(A20) / F: 5′-GAAGAGCAACTGAGATCGAG
R: 5′-GTTGGGATGCTGACACTC / 20
α-actin: alpha smooth muscle actin; β-actin: beta smooth muscle actin; E-selectin; Il-6:interleukin-6; iNOS: incucible nitric oxide synthase; MCP-1: monocyte chemotactic protein-1; MMP-2: matrix metalloproteinase 2; MMP-9: matrix metalloproteinase 9; NF-κB: nuclear factor kappa-b; PCNA: proliferating cell nuclear anitegen; TGF-β: transforming growth facetor beta; TNFAIP3: tumor necrosis factor activated protein 3 (A20);
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