Simvastatin augments revascularization and reperfusion in a murine model of hind limb ischemia – Multimodal imaging assessment

    Simvastatin augments revascularization and reperfusion in a murine model of hind limb ischemia – Multimodal imaging assessment JL Goggi, M Ng, N Shenoy, R Boominathan, P Cheng, S Sekar, KK Bhakoo PII: DOI: Reference:

S0969-8051(16)30224-4 doi: 10.1016/j.nucmedbio.2016.11.007 NMB 7888

To appear in:

Nuclear Medicine and Biology

Received date: Revised date: Accepted date:

2 August 2016 27 October 2016 12 November 2016

Please cite this article as: Goggi JL, Ng M, Shenoy N, Boominathan R, Cheng P, Sekar S, Bhakoo KK, Simvastatin augments revascularization and reperfusion in a murine model of hind limb ischemia – Multimodal imaging assessment, Nuclear Medicine and Biology (2016), doi: 10.1016/j.nucmedbio.2016.11.007

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ACCEPTED MANUSCRIPT Title: Simvastatin augments revascularization and reperfusion in a murine model of hind limb

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ischemia – Multimodal imaging assessment.

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Abbreviated title: Imaging statin effects on neovascularisation

Authors:

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Goggi, JL *a,b; Ng, M a; Shenoy a, N; Boominathan, R a; Cheng, P a; Sekar, S a; Bhakoo, KK a.

a Singapore Bioimaging Consortium, Agency for Science, Technology and Research (A* Star), 11

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Biopolis Way, #07-10 Helios, Singapore, 138667.

b Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore,

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Singapore, 117456.

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*Corresponding author: Dr Julian L Goggi

[email protected] Tel: +65 6478 8901 Fax: +65 6478 8908

Keywords: Statins, integrin, neovascularization, RGD, MRI, ischemia

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ABSTRACT

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Introduction

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Peripheral artery disease can lead to severe disability and limb loss. Therapeutic strategies

amputation rates in progressive PAD.

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focussing on macrovascular repair have shown benefit but have not significantly reduced Proangiogenic small molecule therapies may

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substantially improve limb vascularisation in limb ischemia. The purpose of the current study

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using longitudinal multimodal imaging.

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was to assess the proangiogenic effects of simvastatin in a murine model of hind limb ischemia

Methods

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Mice underwent surgical intervention to induce hind limb ischemia, and were treated with

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simvastatin orally for 28 days. Neovascularisation was assessed using 99mTc-RGD SPECT imaging,

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and macrovascular volume was assessed by quantitative time of flight MRI. At each imaging time point, VEGF expression and capillary vessel density were quantified using immunohistochemical analysis.

Results Simvastatin significantly increased 99mTc-RGD retention in the ischemic hind limb by Day 3 postsurgery, with maximal retention at Day 8. Vascular volume was significantly increased in the ischemic hind limb of simvastatin treated animals, but only by Day 22. Immunohistochemical

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ACCEPTED MANUSCRIPT analysis shows that simvastatin significantly augmented tissue VEGF expression from Day 8 with

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increase in capillary density (CD31+) from Day 14.

Conclusions

99m

Tc-RGD SPECT,

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Early assessment of proangiogenic therapy efficacy can be identified using

which displays significant increases in retention before macrovascular volume changes are

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measureable with MRI.

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Advances in knowledge and implications for patient care Simvastatin offers an effective proangiogenic therapy as an adjunct for management of limb Simvastatin induces integrin expression and vascular remodelling leading to

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ischemia.

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neovascularisation and improved perfusion.

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ACCEPTED MANUSCRIPT INTRODUCTION

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Peripheral artery disease (PAD) is a major cause of disability in a number of pathologies,

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including diabetes and vascular insufficiencies. Primary therapeutic strategies include macrovascular repair or bypass of the blocked vessels [1]. Despite routine clinical use of these

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macrovascular approaches, amputation rates in progressive PAD have remained unchanged

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over the past 30 years [2]. Therapies that induce angiogenesis have been proposed for patients with peripheral ischemic disease as an adjunct to conventional therapies. Angiogenesis is the

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process of forming new blood vessels, sprouting from existing vessels, and is central to normal

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biological processes such as vascular remodelling and wound healing [3, 4]. Hypoxia caused by an ischemic insult can induce limited vascular remodelling spontaneously via the induction of

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vascular endothelial growth factor (VEGF) [5]. Angiogenic therapies are designed to improve

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this remodelling with the aim of re-establishing tissue perfusion. Significant work has concentrated on stimulating angiogenesis to improve perfusion and

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function in ischemic tissues, independent of macrovascular function, using cell based therapies or growth factors; however, efforts have been stymied by safety concerns over side effects, such as hypotension and destabilisation of atherosclerotic plaques [6-11]. Small molecule proangiogenic alternatives based around existing cardiovascular disease therapies could prove to be an effective clinically relevant strategy. Statins, 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibitors, are routinely used clinically to manage cholesterol levels.

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guidelines recommend the use of statins in all patients with PAD due to the high possibility of systemic cardiovascular events [12]. Prophylactic use of statins has also been shown to significantly reduce adverse events in PAD when used to a similar extent as with coronary heart 4

ACCEPTED MANUSCRIPT disease [12-16]. While the mechanism of action is unclear, recent studies have shown that statins not only reduce the risk of adverse events in PAD, but can also improve limb prognosis in

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patients with PAD beyond lipid altering effects alone [12]. Preclinically, statins have been shown to exert proangiogenic effects, exerting protective effects on ischemic injury in the heart

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and promoting angiogenesis in the periphery of animals with normal cholesterol levels [17-20]. Indeed statins have been shown to significantly increase VEGF, eNOS, and angiogenic cytokines

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in preclinical ischemic models in a dose dependent manner [1, 5, 19, 21-23]. Clinically, changes

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in perfusion and functional vascular remodelling after ischemic damage have been assessed using a number of different imaging methodologies; including MR perfusion imaging,

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angiography and laser Doppler imaging. However, these methodologies are optimised for large vessel occlusive disease and fail to differentiate between existing intra-arterial anastomoses

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of angiogenesis.

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and de novo blood vessel formation [24-26], and hence, are inherently less sensitive measures

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New blood vessels have been shown to express high levels of integrins. The integrins αVβ3 and αVβ5 are expressed at low levels on epithelial cells and mature endothelial cells, but are highly expressed on activated endothelial cells during neovasculature [27]. These integrins act as receptors for proteins expressing the arginine-glycine-aspartate (RGD) tripeptide motif [28]. Integrins are attractive targets for angiogenesis imaging, and significant efforts have been made toward the development of ligands for positron emission tomography (PET) and single photon emission computed tomography (SPECT) molecular imaging incorporating the RGD tripeptide motif [29]. Such biomarkers may prove useful for the early monitoring of the efficacy of angiogenic therapies for the treatment of PAD and may help elucidate the temporal 5

ACCEPTED MANUSCRIPT mechanisms of vascular remodeling. In the current study we use multimodal imaging to longitudinally follow vascular recovery and remodelling in a mouse model of hind limb ischemia

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treated with statins over 28 days to determine whether integrin imaging may prove useful for the early monitoring of statin induced angiogenesis and vascular remodeling, than conventional

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single modal imaging techniques.

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MATERIALS AND METHODS

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Animal experiments were performed in accordance with the Institutional Animal Care and Use

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Committee guidelines under ethics number IACUC 120791. Male BALB/c nude mice were

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purchased from In Vivos (Singapore). BALB/c mice were chosen as a model animal as they have been shown to develop the most severe initial ischemia and display relatively poor endogenous

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recovery post arterial occlusion.

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Hind limb ischemia surgery

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The mice were anaesthetized by intraperitoneal injection of ketamine (150 mg/kg) and xylazine (10 mg/kg). An incision of the skin from the medial thigh towards the knee was made and the

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membranes covering the muscle dissected away. Fat tissue was gently pulled towards the

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abdomen and secured. The membranous femoral sheath was pierced to expose the neurovascular bundle and the external iliac artery was gently separated from vein and nerve,

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ligated above the pudendoepigastric trunk proximal to the deep femoral artery twice with 7/0 polypropylene suture (Premilene, Braun, Melsungen AG), and dissected between the two ligations. The incision was closed with 5/0 polypropylene suture (Premilene, Braun, Melsungen AG). The animals received subcutaneous injection of Enrofloxacin (10 mg/kg, once daily) for 5 days and of Buprenorphine (0.1 mg/kg, twice daily) for 3 days post-surgery.

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ACCEPTED MANUSCRIPT Dosing

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Simvastatin (S1792 Selleckchem, USA) was administered immediately after the hind limb

dosed daily by oral gavage for 28 days.

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Preparation of 99mTc-Hynic-RGDfK-dimer (99mTc-RGD)

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ischemia surgery. Subsequently, simvastatin (2.0 mg/kg in saline) or vehicle (saline alone) was

All chemicals were purchased from Sigma-Aldrich Pte. Ltd., unless otherwise specified. HYNIC-

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cyclo(RGDfK) dimer was purchased from ABX advanced biochemical compounds. Na 99mTcO4 was obtained from a 99Mo/99mTc generator (Singapore General Hospital, Department of Nuclear

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Medicine & PET, Radiopharmacy Lab). The radio-HPLC method used a Shimadzu Prominence

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HPLC system equipped with an IN/US systems β-RAM Radio-HPLC detector and Phenomenox C18 Gemini column (4.6 mm x 150 mm). The flow rate was 1.0 mL/min. The mobile phase was a

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gradient from 90% solvent A (Water, 0.05% TFA) and 10% solvent B (Acetonitrile, 0.05% TFA) to

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38% solvent B over 20 min.

Synthesis of 99mTc-Hynic-RGDfK-dimer (99mTc-RGD). To a clean vial were added 50 µL of HYNIC-cyclized RGDfK dimer (1 mg/mL in water), 100 µL of tricine (100 mg/mL in 0.025 M sodium succinate buffer), 20 µL of tin (II) chloride (3.0 mg/mL in 0.1 M HCl solution) and 1.85-2.22 GBq (50-60 mCi) of Na99mTcO4 solution. The reaction solution was incubated at room temperature for 10 minutes, after which was added 3 mg of triphenylphosphine-3,3’,3’’-trisulfonic acid trisodium salt (dissolved in 100 µL of 0.25M sodium succinate buffer, pH 5.0) and the mixture was heated at 99 °C for 30 mins. After the 8

ACCEPTED MANUSCRIPT radiolabelling reaction, an aliquot was analysed by radio-HPLC. In our radiotrace the

99m

Tc-

Hynic-RGDfK-dimer appeared as a double peak (tR= 16 mins). Previous studies have shown that

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the 99mTc-Hynic-RGDfK dimer displays better binding than the monomeric version and that the observed double peak is due to the presence of two diastereomers [30] and that the binding

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affinity of these two diastereomers is equivalent [31].

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Doses preparation for animal Studies.

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The radiolabelled compound was purified using C18-Sep-Pak (Sep-Pak C18 light, 100 mg cartridge) before animal studies. The Sep-Pak C18 cartridge was conditioned with 10 mL of

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acetonitrile followed by washing with 10 mL of water. The radiotracer was loaded onto the SepPak cartridge. The Sep-Pak cartridge was then washed with 0.9% NaCl solution (10 mL) to elute

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unbound 99mTc. 99mTc-RGD was eluted with 1.4 mL of 80% ethanol and collected in 3 fractions of 99m

Tc-

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0.4, 0.6 and 0.4 mL each. The fractions were analysed by radio-HPLC and fractions with

RGD were combined. Ethanol was removed under a stream of nitrogen gas. Doses for animal

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studies were prepared by dissolving the purified radiotracer in 1x phosphate buffered saline solution to obtain a concentration of 150 MBq/mL. The overall radiochemical yield was 49-65%, non-decay corrected over 112-143 min (n=5). The specific activity of

99m

Tc-RGD was 31.2-44.3

GBq/µmol (n=5). The radiochemical purity of the formulated product was above 95%. SPECT imaging with 99mTc-RGD BALB/c nude mice (n = 6 per imaging group) were injected with a solution of

99m

Tc-RGD

(~30 MBq in 0.2 ml) via the lateral tail vein, and the animals imaged under isoflurane

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ACCEPTED MANUSCRIPT anaesthesia (2% alveolar concentration). Biological monitoring for respiration and temperature was performed using a BioVet system (m2m imaging, Cleveland, OH). Small-animal SPECT

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imaging was performed using a nanoSPECT (MEDISO, Hungary). Static images were acquired at 70–90 min post injection (based on prior dynamic imaging studies to optimise SNR; data not

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shown). Low dose CT images (40 kV, 500 µA; 4×4 binning, 200 µm resolution) were acquired for anatomical information. Images were reconstructed using in-built image reconstruction,

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visualization and analysis software supplied by the manufacturer, whilst SPECT and CT data

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were analysed by using Amide software (Sourceforge 10.3, http://amide.sourceforge.net). The SPECT and CT images were co-registered to confirm anatomical location of the

99m

Tc-RGD

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uptake. Uptake of radioactivity was determined by placement of a Region of Interest (ROI) over the muscle of interest delineated using the CT images. The tissue concentrations were

MRI TOF imaging

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measured using ROI analysis and are presented as percent injected dose/gram (%ID/g).

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MRI-Time of Flight imaging was performed using a Bruker 9.4T Biospec MRI scanner. An optimized flow compensated gradient-echo time of flight (TOF) protocol with a spatial resolution of 0.109mm/pixel x 0.109mm/pixel, 0.35mm slice thickness and 150 slices. This protocol was applied at ultra-high field in order to increase the vascular signal relative to surrounding tissues by saturating the stationary tissue signal with very short TR (the longitudinal magnetization does not allow time to recover) and favouring the inflow effect (the signal from the blood flow is made stronger than that of the saturated tissues).

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ACCEPTED MANUSCRIPT MRI TOF image analysis

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MRI TOF data was analysed using FIJI (LOCI MA, WA, USA). The DICOM data was viewed in 2D

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and a spline region of interest used to delineate the entire axial view of the intact right leg as well as the entire axial view of the ischemic left leg above the pudendoepigastric trunk. This 2D

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ROI was extended for 60 slices caudally in the axial plane and great care was taken to exclude

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signal from fat tissue. The imaging data was converted into 8-bit grey-scale and a binary threshold used so that no signal is observable from the surrounding non-vascular tissues. The

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number of pixels above threshold were summed within each ROI and multiplied by the voxel volume to provide a quantitative measurement of the signal within the region of interest. This

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was repeated for a total of 60 slices from the point of ligation above the pudendoepigastric

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trunk and the data summed from each volume of interest.

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Immunohistochemical assessment of capillary vessel number and VEGF expression

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Once assayed for radioactivity, the excised calf and thigh muscle (gastrocnemius, gracilis and adductors) were immediately fixed in neutral buffered formalin. Muscle specimens were fixed and stained for CD31 using anti-CD31 polyclonal antibody (Abcam Singapore Pte Ltd) with a chromagen endpoint, and manually analysed in a masked manner to treatment. Muscle capillary number were calculated as CD31-positive vessel number per 10 fields of view assessed per sample. The muscle specimens were also stained for VEGF using anti-VEGF monoclonal antibody (Abcam Singapore Pte Ltd). Binding was analysed in a masked manner to treatment assignment manually. VEGF expression was calculated as the percentage VEGF-positive cells per 10 fields of view assessed per sample. 11

ACCEPTED MANUSCRIPT RESULTS

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Quantification of limb retention of 99mTc-RGD peptide Small animal SPECT imaging was performed on Days 1, 3, 8, 14 and 28 days post initiation of

in Figure 1a the retention of

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hind limb ischemia. Simvastatin treatment was initiated on the day of surgery. As can be seen Tc-RGD in the ischemic legs, both simvastatin and vehicle

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treated, was significantly higher post-surgical intervention than the intact leg over the time

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course assessed indicating a level of angiogenesis induced by hypoxia. Simvastatin treatment (0.46 ± 0.09%ID/g) had no significant effect on retention of 99m

Tc-RGD compared to vehicle

Tc-RGD was increased significantly

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treatment (0.55 ± 0.13%ID/g) on Day 1. The retention of

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in the simvastatin treated ischemic leg by Day 3 (0.95 ± 0.20%ID/g compared to 0.66±

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0.24%ID/g in the vehicle treated ischemic leg; P<0.05*) and was further increased at Day 8

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(2.01 ± 0.50%ID/g compared to 1.27 ± 0.34%ID/g in the intact leg; P<0.01**, Figure 1a).

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Specificity of binding was shown by blockade with excess cold Hynic-RGDfK dimer (dosed at 30mg/kg IV 5 minutes prior to injection of

99m

Tc-RGD) which abolished the uptake in both the

simvastatin treated ischemic leg (0.46 ± 0.21%ID/g) and the vehicle treated ischemic leg (0.31 ± 0.12%ID/g) at Day 8. By Day 14 the retention of 99mTc-RGD in the simvastatin treated ischemic leg (1.48 ± 0.28%ID/g) was no longer significantly different from the vehicle treated ischemic leg (1.27 ± 0.44 %ID/g). At Day 22 no difference was observed (0.79 ± 0.23%ID/g in the simvastatin treated ischemic leg compared to 0.73 ± 0.40%ID/g in the vehicle treated ischemic leg) nor at Day 28 (0.47 ± 0.15%ID/g in the simvastatin treated ischemic leg compared to 0.59 ± 0.23%ID/g in the vehicle treated ischemic leg). 13

ACCEPTED MANUSCRIPT Assessment of vascular volume using MRI time of flight imaging

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MRI time of flight (TOF) imaging was performed on Days 0, 1, 3, 8, 14, 22 and 28 days post

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initiation of hind limb ischemia. The TOF data show that the vascular volume in the right leg was similar prior to surgery with 5.92 ± 0.42 mm3 and 5.52 ± 0.56 mm3 in the simvastatin and After surgical induction of hind limb ischemia, the

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vehicle treated animals respectively.

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vascular volume in the ischemic leg was significantly reduced from Day 1 onwards (1-way

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ANOVA with Bonferroni post-test) in both the simvastatin treated and vehicle treated animals. As shown in Figure 2, the vascular volume in the simvastatin treated ischemic leg (0.44 ± 0.38

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mm3) was comparable to the vehicle treated ischemic leg (0.32 ± 0.27mm3) on Day 1 immediately post-surgical intervention. The vascular volume increased in both the simvastatin

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and vehicle treated ischemic legs over the time course studied, with 1.10 ± 0.56mm3 and 0.90 ±

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0.75mm3 measured in the simvastatin and vehicle treated ischemic legs respectively on Day 3, and 2.65 ± 1.35mm3 measured in the simvastatin and 2.88 ± 0.94mm3 in the vehicle treated

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ischemic legs on Day 8. A trend toward higher vascular volume was observed on Day 14 in the simvastatin treated animals with the vascular volume of the ischemic leg measuring 3.78 ± 0.95mm3 and the vehicle treated ischemic leg 3.11 ± 1.39 mm3. This trend reached significance at Day 21 with the simvastatin treated ischemic leg measuring 4.55 ± 1.06mm3 (P<0.05) and the vehicle treated ischemic leg 2.91 ± 1.45 mm3. The vascular volume was maintained on Day 28 with simvastatin treated ischemic leg measuring 4.66 ± 1.23mm3, and the vehicle treated ischemic leg 3.86 ± 0.92 mm3.

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ACCEPTED MANUSCRIPT Muscle tissue capillary vessel number analysis

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Muscle tissue was assessed for vessel density ex vivo on each of the imaging days in a separate

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cohort of animals (n=4). Figure 3 shows the number of vessels in the muscle from the ischemic and intact limbs on each day studied, whilst Figure 4 shows a representative field of view from

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ischemic muscle tissue on Day 14. The number of vessels in the simvastatin treated ischemic

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leg (54.5 ± 9.6) was comparable to the vehicle treated ischemic leg (53.2 ± 8.1) on Day 3 postsurgical intervention. The number of CD31 positive vessels increased slightly on Day 8 (89.3 ±

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11.9 measured in the simvastatin and 82.8 ± 17.1 in the vehicle treated ischemic leg). Significantly higher vessel density was observed on Day 14 in the simvastatin treated ischemic

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leg (106.5 ± 18.1) compared to the vehicle treated (80.4 ± 8.4; p<0.05, 1-way ANOVA with post

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hoc Tuckey’s test). This significance was maintained at Day 21 (simvastatin treated ischemic leg

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102.5 ± 15.6 and the vehicle treated ischemic leg 79.0 ± 13.7; p<0.05) and at Day 28 (simvastatin treated ischemic leg 109.8 ± 24.8 and vehicle treated ischemic leg 74.2 ± 8.7;

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p<0.05).

Muscle tissue VEGF level analysis Muscle VEGF expression was also assessed ex vivo on each of the imaging days in a separate cohort of animals (n=4). Figures 5 shows the % area expressing VEGF in the muscle from the ischemic and intact limbs on each of the days studied and Figure 6 shows a representative field of view from ischemic muscle tissue on Day 8. The % area expressing VEGF in the simvastatin treated ischemic leg (32.0 ± 2.9) was comparable to the vehicle treated ischemic leg (26.0 ± 10.1) on Day 3. Significantly higher expression was observed on Day 8 in the simvastatin 15

ACCEPTED MANUSCRIPT treated animals with the % area expressing VEGF in the ischemic leg measuring 64.8 ± 13.9 and the vehicle treated ischemic leg 41.0 ± 16.7 mm3; p<0.05, 1-way ANOVA with post hoc Tuckey’s

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test). This higher expression was no longer significant by Day 14 (simvastatin treated 50.3 ± 11.1 and the vehicle treated 36.6 ± 13.0). Similar areas of expression were noted at the Day 21

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(simvastatin treated 31.3 ± 8.7 and the vehicle treated 27.6 ± 10.0) and Day 28 time points

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(simvastatin treated 33.5 ± 8.8 and the vehicle treated 24.8 ± 11.0).

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ACCEPTED MANUSCRIPT DISCUSSION

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The present study demonstrates the potential for simvastatin to significantly augment

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neovasculature and to modulate remodelling of the vasculature after an ischemic insult. This proangiogenic effect of simvastatin is dependent on the presence of hypoxia caused by

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ischemic insult, as no angiogenesis or vascular remodelling is observed in the intact non-

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ischemic leg. Imaging of neovasculature with 99mTc-RGD displays significantly increased binding to integrins in the injured leg within 3 days of treatment. Ex vivo analysis of the ischemic

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muscle confirms increased expression of the proangiogenic protein VEGF in the simvastatin treated limb at Day 8 post injury with an increasing trend evident from day 3. Previous studies

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have shown that statins augment collateral vasculature growth in response to ischemia by

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enhancing proangiogenic proteins and cytokines in the area of ischemia, and also recovery in

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blood flow in ischemic limbs, followed longitudinally using MRA and laser Doppler perfusion imaging [32, 33]. Interestingly, increases in capillary number were observed ex vivo from day

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14, but improvements in vascular volume in vivo, assessed by MRI, were only observed at day 22 after the ischemic insult. While MRI angiography is used routinely to assess the apparent changes in vascular volume after peripheral limb ischemia, it is significantly affected by vasomotor tone and the presence of pre-existent inter-arterial anastomoses [24].

Thus,

imaging methodologies to assess perfusion or vascular volume alone may not provide an accurate early assessment of the efficacy of proangiogenic therapeutics, but may provide useful information on functional perfusion and recovery of newly induced vessels and collaterals. To our knowledge, this is the first report documenting integrin imaging for the early assessment of the proangiogenic effects of statins in hind limb ischemia. 17

ACCEPTED MANUSCRIPT Clinical guidelines recommend the use of statins in all patients with PAD. While this has been shown to lead to a more favourable limb prognosis, the expected functional improvement

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induced by vascular remodelling is not evident [12, 14]. This may be due to the fact that the prescribed dose required for lipid altering effects is not optimal for proangiogenic effects.

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Although the precise mechanism by which statins exert their effects on angiogenesis and vasculogenesis is unknown, it seems that their effects are pleiotropic, with statins displaying

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proangiogenic or angiostatic effects are dependent on the dose [34]. The proangiogenic effects

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are likely through HMG-CoA reductase inhibition of mevalonate production, which is known to inhibit PI3K. Active PI3K leads to an increase in Akt phosphorylation by PDK-1 kinase, and

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subsequently phosphorylation and activation of endothelial nitric oxide synthase leading to angiogenesis and vasculogenesis [5, 18, 20, 22, 32, 35]. The angiostatic effects seem to be

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dependent on protein prenylation [36]. Indeed, simvastatin has been shown to reduce VEGF

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expression and angiogenesis at high concentrations in a model of hypercholesterolemia [37]

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but promote angiogenesis in ischemic models at lower doses [17, 38, 39].

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SUMMARY

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PAD is a very common condition for which there is currently no effective drug therapy. Statins

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have been proposed as small molecule therapies to improve angiogenesis and limb perfusion.

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In our present study, we document an improvement in angiogenesis by integrin imaging after 3 days of treatment, and by MRI after 22 days. The in vivo imaging results are supported by

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immunohistochemistry analysis, showing higher VEGF levels after 8 days and an increase in

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capillary density after 14 days. These results suggest that the use of simvastatin at the correct

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dose may benefit revascularisation in PAD patients.

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Conflict of interest

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The authors declare no conflict of interest.

Source of funding

This work was supported by funding from the Singapore Bioimaging Consortium, A*STAR.

Disclosures None.

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[16] Poredos P and Jezovnik MK. Dyslipidemia, statins, and venous thromboembolism. Seminars in thrombosis and hemostasis 2011;37:897-902. [17] Thirunavukkarasu M, Selvaraju V, Dunna NR, Foye JL, Joshi M, Otani H, et al. Simvastatin treatment inhibits hypoxia inducible factor 1-alpha-(HIF-1alpha)-prolyl-4-hydroxylase 3 (PHD-3) and increases angiogenesis after myocardial infarction in streptozotocin-induced diabetic rat. International journal of cardiology 2013;168:2474-80. [18] Skaletz-Rorowski A, Kureishi Y, Shiojima I, and Walsh K. The pro- and antiangiogenic effects of statins. Seminars in vascular medicine 2004;4:395-400. [19] Skaletz-Rorowski A and Walsh K. Statin therapy and angiogenesis. Current opinion in lipidology 2003;14:599-603. [20] Orbay H, Hong H, Koch JM, Valdovinos HF, Hacker TA, Theuer CP, et al. Pravastatin stimulates angiogenesis in a murine hindlimb ischemia model: a positron emission tomography imaging study with (64)Cu-NOTA-TRC105. American journal of translational research 2013;6:5463. [21] Li Y, Zhang D, Zhang Y, He G, and Zhang F. Augmentation of neovascularization in murine hindlimb ischemia by combined therapy with simvastatin and bone marrow-derived mesenchymal stem cells transplantation. Journal of biomedical science 2010;17:75. [22] Weis M, Heeschen C, Glassford AJ, and Cooke JP. Statins have biphasic effects on angiogenesis. Circulation 2002;105:739-45. [23] Katsumoto M, Shingu T, Kuwashima R, Nakata A, Nomura S, and Chayama K. Biphasic effect of HMG-CoA reductase inhibitor, pitavastatin, on vascular endothelial cells and angiogenesis. Circulation journal : official journal of the Japanese Circulation Society 2005;69:1547-55. [24] Jacoby C, Boring YC, Beck A, Zernecke A, Aurich V, Weber C, et al. Dynamic changes in murine vessel geometry assessed by high-resolution magnetic resonance angiography: a 9.4T study. Journal of magnetic resonance imaging : JMRI 2008;28:637-45. [25] Brenes RA, Jadlowiec CC, Bear M, Hashim P, Protack CD, Li X, et al. Toward a mouse model of hind limb ischemia to test therapeutic angiogenesis. Journal of vascular surgery 2012;56:1669-79; discussion 79. [26] Luo Y, Mohning KM, Hradil VP, Wessale JL, Segreti JA, Nuss ME, et al. Evaluation of tissue perfusion in a rat model of hind-limb muscle ischemia using dynamic contrast-enhanced magnetic resonance imaging. Journal of magnetic resonance imaging : JMRI 2002;16:277-83. [27] Carmeliet P and Collen D. Molecular basis of angiogenesis. Role of VEGF and VE-cadherin. Annals of the New York Academy of Sciences 2000;902:249-62; discussion 62-4. [28] Battle MR, Goggi JL, Allen L, Barnett J, and Morrison MS. Monitoring tumor response to antiangiogenic sunitinib therapy with 18F-fluciclatide, an 18F-labeled alphaVbeta3-integrin and alphaV beta5-integrin imaging agent. Journal of nuclear medicine : official publication, Society of Nuclear Medicine 2011;52:424-30. [29] Chen H, Niu G, Wu H, and Chen X. Clinical Application of Radiolabeled RGD Peptides for PET Imaging of Integrin alphavbeta3. Theranostics 2016;6:78-92. [30] Janssen M, Oyen WJ, Massuger LF, Frielink C, Dijkgraaf I, Edwards DS, et al. Comparison of a monomeric and dimeric radiolabeled RGD-peptide for tumor targeting. Cancer biotherapy & radiopharmaceuticals 2002;17:641-6.

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[31] Liu S, Edwards DS, Ziegler MC, Harris AR, Hemingway SJ, and Barrett JA. 99mTc-labeling of a hydrazinonicotinamide-conjugated vitronectin receptor antagonist useful for imaging tumors. Bioconjugate chemistry 2001;12:624-9. [32] Sata M, Nishimatsu H, Osuga J, Tanaka K, Ishizaka N, Ishibashi S, et al. Statins augment collateral growth in response to ischemia but they do not promote cancer and atherosclerosis. Hypertension 2004;43:1214-20. [33] Zhang Y, Zhang R, Li Y, He G, Zhang D, and Zhang F. Simvastatin augments the efficacy of therapeutic angiogenesis induced by bone marrow-derived mesenchymal stem cells in a murine model of hindlimb ischemia. Molecular biology reports 2012;39:285-93. [34] Weiss CO and Varadhan R. Risk-treatment paradox in use of statins. Jama 2004;292:169; author reply [35] Izumi Y, Shiota M, Kusakabe H, Hikita Y, Nakao T, Nakamura Y, et al. Pravastatin accelerates ischemia-induced angiogenesis through AMP-activated protein kinase. Hypertension research : official journal of the Japanese Society of Hypertension 2009;32:675-9. [36] Park HJ, Kong D, Iruela-Arispe L, Begley U, Tang D, and Galper JB. 3-hydroxy-3methylglutaryl coenzyme A reductase inhibitors interfere with angiogenesis by inhibiting the geranylgeranylation of RhoA. Circulation research 2002;91:143-50. [37] Wilson SH, Herrmann J, Lerman LO, Holmes DR, Jr., Napoli C, Ritman EL, et al. Simvastatin preserves the structure of coronary adventitial vasa vasorum in experimental hypercholesterolemia independent of lipid lowering. Circulation 2002;105:415-8. [38] El-Azab MF, Hazem RM, and Moustafa YM. Role of simvastatin and/or antioxidant vitamins in therapeutic angiogenesis in experimental diabetic hindlimb ischemia: effects on capillary density, angiogenesis markers, and oxidative stress. European journal of pharmacology 2012;690:31-41. [39] Koksoy C, Ozis E, Cakmak A, Yazgan U, Okcu-Heper A, Koksoy A, et al. Simvastatin pretreatment reduces the severity of limb ischemia in an experimental diabetes model. Journal of vascular surgery 2007;45:590-6.

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Tc-RGD measured by longitudinal SPECT imaging

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Figure 1a. Graph showing retention of

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(~30 MBq, acquired from 70-90 mins post injection under isoflurane anaesthesia). Retention was significantly higher in the simvastatin treated ischemic limb compared to the vehicle

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treated ischemic limb on Days 3 and 8 post ligation (n=7 (n=3 blocked), *P<0.05, **P<0.01, 1-

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way ANOVA with post hoc Tuckey’s test, data shown as %ID/g ± SEM).

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Figure 1b. Representative images displaying retention of

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ischemic limb (left column) compared to the simvastatin treated ischemic limb (right column)

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on each of the imaging days post ligation.

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Figure 2. Graph showing hind limb vascular volume measured by TOF MRI. Vascular volume was significantly higher in the simvastatin treated ischemic limb compared to the vehicle treated

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ischemic limb on Day 22 post ligation (n=7, *P<0.05, 1-way ANOVA with post hoc Tuckey’s test, mean volume in mm3 ± SEM).

Figure 3. Graph showing the number of CD31 positive vessels in hind limb vascular volume measured by immunohistochemical assessment of anti-CD-31 antibody staining. The number of CD31 positive capillaries was significantly higher in the simvastatin treated ischemic limb compared to the vehicle treated ischemic limb from Day 14 post ligation (n=4, *P<0.05, 1-way ANOVA with post hoc Tuckey’s test, mean number of capillaries ± SEM).

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treated ischemic muscle (A) and simvastatin treated ischemic muscle (B).

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Figure 5. Graph showing the percentage area of VEGF positive staining in hind limb muscle measured by immunohistochemical assessment of anti-VEGF antibody staining. VEGF staining

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treated ischemic limb at Day 8 post ligation (n=4, *P<0.05, 1-way ANOVA with post hoc

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Tuckey’s test, mean % area stained ± SEM).

Figure 6. Representative muscle sections taken at Day 8 displaying staining for VEGF in vehicle

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treated ischemic muscle (A) and simvastatin treated ischemic muscle (B).

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Figure 6

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