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Simvastatin Supplemental methods Page

Supplemental Methods and materials

Experimental animals

Appropriate Institutional Animal Care and Use Committee approval was obtained prior to performing any procedures. The housing and handling of the animals was performed in accordance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals revised in 2000 1. Animals were housed at 22°C temperature, 41% relative humidity, and 12-/12-hour light/dark cycles. Animals were allowed access to water and food ad libitum. Anesthesia was achieved with intraperitoneal injection of a mixture of ketamine hydrochloride (0.20 mg/g) and xylazine (0.02 mg/g) and maintained with intraperitoneal pentobarbital (20-40 mg/kg). Seventy-nine male C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME) weighing 25-30 grams were used for the present study as depicted (Fig. 1). Chronic kidney disease was created by surgical removal of the right kidney accompanied by ligation of the arterial blood supply to the upper pole of the left kidney as described previously (Fig. 1A) 2. Three weeks after nephrectomy, the animals were started on simvastatin (40 mg/kg administered IP three times per week) or PBS (equal amount of volume used for simvastatin I.P. controls). Simvastatin was prepared as described else where 3. A week later, an AVF was created by connecting the right carotid artery to the ipsilateral jugular vein (Fig. 1B) 2, 4. Animals were sacrificed at day 7, day 14, and day 28 following AVF placement for either real time polymerase chain reaction (RT-PCR), protein, or histomorphometric analyses (Fig. 1C). Serum BUN and creatinine were measured by removing blood from the tail vein at baseline (before nephrectomy), at AVF creation, and at the time of sacrifice. A separate group of

mice that did not undergo nephrectomy had an AVF placed. These animals were started on simvastatin (40 mg/kg I.P.) or PBS (equal amount of volume I.P.) every other day one week before AVF placement and then sacrificed 4-weeks after fistula placement for histomorphometric analysis.

Tissue processing and immunohistochemistry

Each outflow vein from each animal was embedded in paraffin length-wise so that the sections would be orthogonal to the long axis of the vessel (Fig. 1B). Typically, 80 to 120, 4-mm sections were obtained and the cuff used to make the anastomosis could be visualized. Every 40-mm, 2-4 sections were stained with hematoxylin and eosin, KI-67, a-SMA, hypoxyprobe, HIF-1 a, smoothelin, smooth muscle heavy chain (SMHC), picrosirius red, or TUNEL. Cellular proliferation was determined by staining for Ki-67 on sections removed from the outflow vein by performing quantification at different time points. Smooth muscle density was determined by staining for α-SMA on sections removed from the outflow vein by performing quantification at the different time points. Immunohistochemistry for Ki-67 and α-SMA were performed on paraffin-embedded sections from the outflow vein after transfection with either simvastatin or control groups using the EnVision (Dako, Carpinteria, CA) method with a heat-induced antigen retrieval step 5, 6. The following antibodies were used: mouse monoclonal antibody Ki-67(DAKO, Carpentaria, CA; 1:400), mouse monoclonal HIF-1a (Novus Biologicals, Littleton, CO; 1:500), rabbit polyclonal antibody to mouse for α-SMA (Abcam, Cambridge, MA; 1:400), rabbit polyclonal antibody to mouse for VEGF-A (Abcam, Cambridge, MA; 1:800), monoclonal antibody to mouse for MMP-2 (Abcam, Cambridge, MA; 1:400), rabbit polyclonal antibody to mouse for MMP-9 (Abcam, Cambridge, MA; 1:800), mouse monoclonal to smooth muscle heavy chain (Novus Biologicals; 1:500), or smoothelin (Santacruz Biotech, Santa Cruz, CA; 1:500). IgG antibody staining was performed to serve as controls.

The outflow vein with the cuff anastomosis was harvested as shown (Fig. 1B) and then embedded. In this model, the stenosis forms at the outflow vein and this can be identified easily as the cuff used to create the anastomosis is a landmark. An average of 12 multiple contiguous serial four-micrometer sections were stained with hematoxylin and eosin and analyzed for histomorphometric analyses (see later).

Hypoxyprobe staining at day 14 and 28

We assessed hypoxic changes in the outflow vein after treatment with either simvastatin or controls using hypoxyprobe using HypoxyprobeTM-1 (a substituted derivative of pimonidazole hydrochloride). HypoxyprobeTM-1 upon activation forms stable covalent adducts with thiol groups of proteins, peptides and amino acids of hypoxic tissue. Mice were injected with 60 mg/kg HypoxyprobeTM-1 i.p. (EMD Millipore, Billerica, MA). Thirty minutes following injection, mice were sacrificed and outflow veins were dissected and fixed as specified for histological analysis. Four-micrometer paraffin embedded sections were stained with the anti-hypoxyprobe-1 Ab as per manufacturer’s directions.

TUNEL Staining at day 14 and 28

TUNEL staining was performed on paraffin-embedded sections from the outflow vein after transfection with either simvastatin or controls as specified by the manufacturer (DeadEnd Colorimetric tunnel assay system, G7360, Promega).

Picrosirius red staining at day 14 and 28

The paraffin embedded sections were de-waxed and hydrated before being stained with picrosirius red for one hour to achieve a near-equilibrium staining. The sections were then washed twice with acidified distilled water before being subjected to dehydration process in sequential grades of alcohol before being mounted in a resinous medium.

SDS PAGE Zymography for MMP-2 and MMP-9

MMP-2 and MMP-9 protein activities were determined using zymographic analysis. This was performed on homogenates from cultured cells or outflow veins treated with simvastatin or control as described previously 5, 6.

RNA isolation

The tissue was stored in RNA stabilizing reagent (Qiagen, Gaithersburg, MD) as per the manufactures guidelines. To isolate the RNA, the specimens were homogenized and total RNA isolated using RNeasy mini kit (Qiagen) 2, 4.

Real time polymerase chain reaction (RT-PCR) analysis

Expression for the gene of interest was determined using RT-PCR analysis 4. Briefly, first-strand complementary DNA (cDNA) was synthesized using superscript III first strand (Invitrogen, Carlsbad, CA) according to the manufacturer's guidelines. cDNAs specific for the genes analyzed were amplified using commercial primers purchased from SA Biosciences (Frederick, MD). PCR products were analyzed on 1.5% (w/v) agarose gels containing 0.5-µg/ml ethidium bromide. Bands were quantified by scanning densitometry (Image J version 1.43, NIH, Bethesda, MD). An area of the gel image that was devoid of signal was assigned to be the background value. Then each band representing the gene of interest was analyzed for the density above background. Next, it was normalized to the amount of loading of mRNA to 18S gene to ensure that there were no differences in loading and then pooled for all the animals in the different treatment groups for each time period 2, 4.

Hypoxia chamber

One hundred thousand of NIH 3T3 cells were treated with either simvastatin or controls and then made hypoxic for 8 or 24 hours as previously described 7.

Proliferation Assay

100,000 NIH 3T3 cells treated with either simvastatin or controls and then seeded in 24-well plates and cultured for 24 h in DMEM medium. After 20 h, 1 mCi of (3H) thymidine was added to each well; 4 hrs later, cells were washed with chilled PBS, fixed with 100% cold methanol and collected for measurement of trichloroacetic acid-precipitable for radioactivity. Experiments were repeated at least three times for each time point.

Cell Migration Assay

One hundred thousand NIH 3T3 cells were treated with either simvastatin or controls were seeded in 8-micron trans-wells, pre-coated with low growth factor matrigel in a serum free media. The complete media was supplemented under the trans-well and incubated for 6 h at 37oC. After 6h, trans-wells were washed with PBS and fixed with paraformaldehye (4% v/v). Finally trans-wells were stained with bromophenol (0.1%) solution. The cells from upper side were removed with cotton tip applicators. The cells at bottom side were counted for analysis.

Caspase 3 Activity

Apoptosis was assessed using an ELISA assay for caspase 3. Cellular protein was extracted from cultured cells. The enzymatic activity of caspase 3 was accessed by Caspase Glo assay (G811C, Promega, Madison, WI).

Morphometry and Image Analysis

Sections immunostained for hematoxylin and eosin stains were viewed with an Axioplan 2 Microscope (Zeiss, Oberkochen, Germany) equipped with a Neo-Fluor × 20/0.50 objective and digitized to capture a minimum of 3090 × 3900 pixels using a Axiocam camera (Zeiss) 5, 6. Images covering one entire cross-section from each section of the outflow vein treated with simvastatin or controls were acquired and analyzed using KS 400 Image Analysis software (Zeiss). Ki-67 (brown), a-SMA positive (brown), smooth muscle heavy chain (brown), smoothelin (brown), TUNEL positive (brown), HIF-1a (brown), or hypoxyprobe (brown) were highlighted, in turn, by selecting the appropriate RGB (red-green-blue) color intensity range and then counted. The color intensity was adjusted for each section to account for decreasing intensity of positive staining over time. This was repeated twice to ensure intraobserver variability was less than 10%. Sections were subsequently viewed with an Axioplan 2 Microscope (Zeiss) equipped with a Neo-Fluor × 20/0.50 objective and digitized to capture at least 1030 × 1300 pixels and cell density determined along with the vessel wall and luminal vessel areas. The area was measured by tracing the vessel wall using an automated program 6.

Statistical methods

Data are expressed as mean ± SEM. Multiple comparisons were performed with two-way ANOVA followed by Student t-test with post hoc Bonferroni’s correction. Because of the Bonferroni correction, significant difference from control value was indicated by *P < 0.01, **P < 0.001, or #P < 0.0001. SAS version 9 (SAS Institute Inc., Cary, N.C.) was used for statistical analyses.


Figure legends:

Supplementary Fig. 1: Hematoxylin and eosin (H and E) staining of the simvastatin treated vessels showing reduced venous neointimal hyperplasia and positive vascular remodeling in animals with AVF and normal kidney function at day 28. (A) is the representative sections after hematoxylin and eosin (H and E) staining at the venous stenosis of either control (C) or simvastatin (SV) at 28 days after AVF placement. The upper panels are 10X and the lower panels are 40X. L is the lumen, NI is the neointima, and ADV is the adventitia/media. (B) is the semiquantitative analysis which shows a significant decrease in the average area of the neointima of the simvastatin treated vessels (SV) when compared to control (C) group (P<0.05). (C) is the semiquantitative analysis which shows no difference in the average area of the media/adventitia of the simvastatin treated vessels (SV) when compared to control (C). (D) demonstrates a significant increase in the average lumen vessel area of the simvastatin treated vessels (SV) when compared to control (C) group (P<0.05). (E) shows a significant decrease in the average cell density in the neointima of the simvastatin treated vessels (SV) when compared to control (C) group (P<0.01). (F) shows similar results in that there is a significant reduction in the average cell density in the media/adventitia of the simvastatin treated vessels (SV) when compared to control (C) group (P<0.001). Each bar represents mean + SEM of 3-5 animals. Significant differences between simvastatin treated and controls is indicated by *P<0.01 or **P<0.001.

References:

1. Committee on care and use of laboratory animals of the institute of laboratory animal resources. Government print office: Washington, DC, 1996.

2. Misra S, Shergill U, Yang B, et al. Increased expression of HIF-1alpha, VEGF-A and its receptors, MMP-2, TIMP-1, and ADAMTS-1 at the venous stenosis of arteriovenous fistula in a mouse model with renal insufficiency. J Vasc Interv Radiol 2010; 21: 1255-1261.

3. Wilson SH, Simari RD, Best PJ, et al. Simvastatin preserves coronary endothelial function in hypercholesterolemia in the absence of lipid lowering. Arterioscler Thromb Vasc Biol 2001; 21: 122-128.

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6. Misra S, Fu AA, Puggioni A, et al. Increased shear stress with up regulation of VEGF-A and its receptors and MMP-2, MMP-9, and TIMP-1 in venous stenosis of hemodialysis grafts Am J Physiol Heart Circ Physiol 2008; 294: H2219-2230.

7. Misra S, Fu AA, Misra KD, et al. Hypoxia-induced phenotypic switch of fibroblasts to myofibroblasts through a matrix metalloproteinase 2/tissue inhibitor of metalloproteinase-mediated pathway: implications for venous neointimal hyperplasia in hemodialysis access. J Vasc Interv Radiol 2010; 21: 896-902.