Towards a more relevant hind limb model of muscle ischaemia

Atherosclerosis 227 (2013) 1e8

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Review

Towards a more relevant hind limb model of muscle ischaemia Shamim Lotfi a, Ashish S. Patel a, Katherine Mattock a, Stuart Egginton b, Alberto Smith a, Bijan Modarai a, * a

Academic Department of Surgery, Cardiovascular Division, King’s College London, BHF Centre of Excellence, NIHR Biomedical Research Centre at Guy’s and St Thomas’ NHS Foundation Trust, 1st Floor North Wing, St Thomas’ Hospital, London SE1 7EH, United Kingdom b Angiogenesis Research Group, Centre for Cardiovascular Sciences, Medical School, University of Birmingham, United Kingdom

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 July 2012 Received in revised form 24 October 2012 Accepted 24 October 2012 Available online 2 November 2012

Critical limb ischaemia is a severe manifestation of peripheral arterial disease characterised by intractable pain and tissue gangrene. Conventional treatments include percutaneous angioplasty and surgical bypass but up to one third of patients are not amenable to these interventions and will ultimately require amputation. Therapeutic neovascularisation has been proposed as an alternative treatment in these ‘no option’ patients and both cytokines and cells have shown impressive efficacy in the laboratory. Clinical trials in man, however, have had modest results. This discrepancy has put into question the relevance of the pre-clinical assays that are used to test potential agents. One of the most widely used of these assays is the hind limb ischaemia model that is often performed in young, healthy animals. This review critiques the techniques used to induce and assess ischaemia in this model and outlines the reasons why healthy rodents cannot fully recapitulate critical limb ischaemia in aged patients. Strategies that may produce a hind limb model that better simulates the human condition are proposed. Ó 2012 Elsevier Ireland Ltd. All rights reserved.

Keywords: Therapeutic angiogenesis Angiogenesis Hind limb ischaemia Peripheral arterial disease Critical limb ischaemia

Contents 1. 2. 3. 4. 5. 6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 The hind limb ischaemia model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2 Arterial ligation and operative technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2 Assessment of blood perfusion in the ischaemic hind limb . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4 Characteristics related to the animal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7 Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

1. Introduction Peripheral arterial disease affects up to 20% of people over the age of 70 [1]. The build-up of atherosclerotic plaque can cause a severe restriction to flow in the lower limb arteries which in turn leads to critical limb ischaemia (CLI), characterised by intractable pain, ulcers and gangrene. Up to a third of CLI patients are not amenable to conventional interventions such as percutaneous angioplasty or surgical bypass and will ultimately require amputation of the limb [2]. Therapeutic neovascularisation has been * Corresponding author. Tel.: þ44 (0) 2071880216; fax: þ44 (0) 2079288742. E-mail address: [email protected] (B. Modarai). 0021-9150/$ e see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.atherosclerosis.2012.10.060

proposed as an alternative treatment strategy in these ‘no option’ patients [2,3]. This treatment aims to stimulate angiogenesis (growth of capillaries) and arteriogenesis (remodelling of existing arteriolar connections into larger calibre collateral vessels), both of which are important components of the physiological response to reduced tissue perfusion [4e6]. In a minority of patients, this inherent physiological response can restore blood flow and ameliorate lower limb ischaemia, but the majority are unable to mount an appropriate response to vessel occlusion and require the delivery of therapeutic agents to stimulate neovascularisation of the ischaemic muscle. Over the past 20 years, growth factors and cells have been used to promote neovascularisation of ischaemic limbs with the aim of

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improving tissue perfusion sufficiently to prevent tissue necrosis and amputation [1,3]. Angiogenic factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) were used in the initial studies, but there was little evidence of a lasting therapeutic benefit in patients. The focus of treatments aimed at ameliorating ischaemic muscle has shifted to using cells with angiogenic properties, including bone marrow derived mononuclear cells, peripheral blood mononuclear cells and umbilical cord blood-derived mesenchymal stem cells [2]. While in vitro and in vivo pre-clinical studies of these agents and cells have promised clinical efficacy, their long-term benefit evaluated through clinical trials have been modest [1,2,7]. To date no therapeutic agent promoting neovascularisation has been approved for clinical use in CLI patients despite having demonstrated impressive efficacy in the laboratory. This has led some investigators to question the validity of the pre-clinical assays, including the models of angiogenesis and ischaemia that have been used for proof of principle and efficacy testing [2]. Pre-clinical strategies for testing the efficacy of prospective agents include both in vitro and in vivo assays. The endothelial cell tubule formation assay is an example of an in vitro assay that, compared with in vivo assays, is cost effective, technically less demanding, amenable to high throughput analysis and allows testing of human-derived cells, making it especially useful for screening of potential therapeutic agents [8]. An in vivo model, however, incorporates a number of complexities that renders it more representative of the human condition; skeletal muscle vasculature is a 3-dimensional network consisting of capillaries and larger blood vessels comprising a variety of cell types including endothelial cells (EC), smooth muscle cells, pericytes, and monocyte/macrophages that interact with the extracellular matrix. Neovascularisation involves a complex interplay of all of these elements within this microenvironment [8,9]. Rendering the limb of an animal ischaemic is currently considered to be the most effective method for simulating the conditions found in CLI, where local circulatory haemodynamics (i.e. blood flow and shear stress) influence the response generated within the limb following ischaemia as well as after delivery of therapeutic agents.

2. The hind limb ischaemia model The hind limb ischaemia model (HLIM) involves acute interruption of arterial supply and remains the most commonly used pre-clinical in vivo method of assessing the angiogenic and arteriogenic potential of agents and cells [2,10]. The HLIM has been used extensively as an in vivo assay to gain pre-clinical mechanistic and therapeutic insights into revascularisation of ischaemic muscle. The angiogenic and arteriogenic potential of various cytokines such as VEGF [11], viral-based gene therapy approaches [12e14], nanoparticle-drug delivery systems [15], mononuclear cells and stem cells [16e19] have been tested. The HLIM has also been used to assess the ameliorating effect of mechanical interventions such as extracorporeal shock wave therapy and pulsed electromagnetic field magnet on tissue ischaemia [20,21]. The rodent is most frequently used although rabbit, porcine, canine and primate models have also been described [22e25]. The use of small animals such as rodents (in particular the mouse) has the advantage of a wide availability of transgenic strains and lower cost of experimentation, whereas larger animals, such as the pig, more closely mimic the size and haemodynamics of the human vasculature allowing easier identification of blood vessels and the ability to assess blood flow within individual vessels [6]. Although the HLIM is considered to be most clinically relevant to peripheral arterial disease, and especially CLI, it does not entirely reproduce the complex human condition and there are important limitations that must be taken into consideration when interpreting results. 3. Arterial ligation and operative technique The approach used to induce ischaemia determines how closely the HLIM represents the condition it is simulating. In rodents, a 5 mm longitudinal incision is made at the origin of the inguinal crease and the subcutaneous tissue is dissected to expose the anatomical landmarks illustrated in Fig. 1 and the femoral artery and vein are separated by blunt dissection. The femoral artery is then ligated with a single ligature just distal to the origin of the profunda femoris to create ischaemia (Fig. 2) [26]. This method, however, leaves most of the collateral circulation to the lower limb

Fig. 1. (a) Vasculature of the mouse hind limb. (b) Magnified view of the arterial branches in the left groin.

S. Lotfi et al. / Atherosclerosis 227 (2013) 1e8

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Fig. 2. (aed) The technique of inducing hind limb ischaemia in a mouse. The proximal and distal ends of the femoral artery and the proximal profunda femoris artery in the groin are dissected and ligated. The intervening segments are then excised (e) Laser Doppler image following induction of left leg ischaemia showing absence of flow in comparison with the right limb.

intact and consequently blood flow to the limb is fully restored within 7 days [27]. Alternatively, a second ligature can be placed 5 mm below the first and the intervening segment of femoral artery excised to produce more severe ischaemia. Double ligation and excision of a segment of femoral artery in this manner removes the collateral bed, which means that angiogenesis as well as arteriogenesis is required to restore flow. Under these conditions only a third of the original blood flow to the limb is restored 7 days after induction of ischaemia. Concurrent femoral artery excision and ligation of the profunda femoris and circumflex branches increases the severity of ischaemia even further [27] and may be useful in species or strains that have extensive pre-existing collateral circulation (such as C57BL/6 mice) [26]. Simultaneous excision of the external iliac artery and vein, femoral artery and its branches and the femoral vein has also been described [13]. Proximal, multi-level arterial disease (involving iliac and femoral arteries) results in more severe symptoms of ischaemia compared with more distal disease in man but such multi-level arterial ligation in the rodent results in rapid necrosis and auto amputation of the limb and is, therefore, too severe to use as a representative assay for the study of CLI. Conversely, ligation of the femoral artery with a single ligature as described without excision of a segment of artery produces only a mild ischaemia that is akin to intermittent claudication rather than CLI in man [28]. Ligation of the femoral artery at two points and excision of the intervening segment seems to be the most appropriate method for induction of ischaemia, producing signs in the mouse that are similar to those observed in human CLI. Some investigators routinely ligate both the femoral vein and femoral artery to induce hind limb ischaemia. This cannot be considered representative of human CLI since these patients do not generally present with concurrent arterial and venous occlusions. Simultaneous ligation of the femoral vein may also confound any results observed as this significantly inhibits collateral vessel formation in the mouse when compared with femoral artery ligation alone. The femoral vein is in close proximity to the femoral artery in the rodent and dissecting one away from the other can be technically challenging but most investigators who are carefully assessing the

contribution of recruitment of collateral arteries do not ligate both vessels [13]. Hind limb ischaemia is induced by acute ligation or excision of the artery of interest. In man, however, CLI generally occurs as a result of a chronic process associated with the build-up of atherosclerotic plaque over many years leading to arterial stenosis. Studies comparing acute induction of hind limb ischaemia (by femoral artery excision) with gradual occlusion using an ameroid constrictor have found very different responses in the animal [29e 31]. In acute ischaemia blood flow recovery peaked at 84% of normal after 35 days and the gastrocnemius muscle showed significant necrosis. This form of ischaemia appears to be more representative of acute limb ischaemia in man because it is more severe and the pattern of the ulceration and necrosis observed is different from the tissue loss seen in human CLI, which is usually confined to the foot [6,31]. In the chronic ischaemia group, muscle necrosis was not observed and the maximal blood flow recovery was only 68% of normal [31]. It appears that gradual induction of ischaemia simulates the slow, progressive onset of CLI in man, and as such may serve as a more clinically relevant model. Gradual arterial occlusion produces smaller pressure gradients across the collateral arterial bed and consequently expression of shear stress inducible factors such as eNOS and PDGF is attenuated and this leads to reduce collateral vessel recruitment and expansion [31]. The more complete recovery after acute ischaemia may also result from higher expression of SDF-1 alpha in the acutely ischaemic muscle. While some studies assume ischaemic repair is essentially an inflammatory response with muscles showing damage, we and others, who have avoided significant venous trauma or neuropathy, find little evidence of changes in the muscle (macrophages, oedema, centrally located nuclei etc.) [32] unless the ischaemic muscle is made to work (in our case, by electrical stimulation) [10]. It is also feasible that inflammatory cytokines such as interleukin-6 that are abundant after acute induction of ischaemia may blunt the angiogenic response in an otherwise pro-angiogenic environment [33]. This has important implications for the pre-clinical testing of novel therapeutics, the majority of which are carried out using the standard method of acute femoral artery ligation.

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4. Assessment of blood perfusion in the ischaemic hind limb Blood perfusion of the hind limb is commonly assessed using Laser Doppler Perfusion Imaging (LDPI) (Table 1, Fig. 2). Laser light, directed by the LDPI device at the tissue under study is scattered by moving blood within the tissue causing a change in the wavelength and frequency of the light that is reflected back, which is then used to calculate the distribution of velocity of blood cells in the tissue. Unilateral excision of the femoral artery allows the contralateral limb to be used as a control comparator, although changes in gait or recruitment of spinal reflexes mean that the contralateral muscle is not fully equivalent to untreated controls [34,35]. The LDPI is noninvasive and measurements are reproducible [36] thus allowing longitudinal analysis of the effect of the therapeutic agent of interest. Assessment by LDPI is, however, a measure of blood flux

rather than perfusion per se and valid comparisons of measurements taken at different time points relies on ensuring that ambient conditions such as temperature and activity of the animal immediately prior to taking the measurement have been standardised, as LDPI is affected by vasomotor tone. The plantar sole region may be the most reliable area for assessing limb perfusion [26]. This area is hairless and does not require depilation, therefore avoiding the skin irritation and changes in LDPI signal associated with hair removal. The relatively smaller area of interest requires a shorter scan time, which minimises the inconsistencies associated with motion artefact when larger areas such as the thigh are scanned. Gross hind limb perfusion can be estimated invasively with the use of calibrated flow probes around a supply artery, but this method requires careful calibration of the probe and it lacks any spatial resolution to identify regions of ischaemia within the limb [37]. Intra-

Table 1 Blood flow and perfusion assessment techniques. Technique

Description

Advantages

Disadvantages

Laser Doppler Perfusion Imaging (LDPI)

Moving red blood cells create a Doppler shift signal when laser light reaches the skin. The signal is proportional to blood flow and hence used as a surrogate measure of perfusion.

    

Non-invasive Reproducible Real-time capability Measures micro-circulatory changes Allows longitudinal acquisition of data

 Motion-artefact noise  Indirect measure of perfusion  Arbitrary values which necessitates a comparator limb or serial imaging  Perfusion signal varies with tissue property

Arterial flow probes

Probe is fitted around a vessel of interest. Probe contains two ultrasonic transducers opposing a reflective plate. Doppler shift in ultrasound signal is used to calculate velocity across a defined distance and hence flow.

 Real-time flow measurements  Ability to assess waveform  Allows longitudinal acquisition of data

 Invasive  Technically challenging  Only measures gross limb flow and not micro-circulation  Lacks spatial resolution to assess tissue ischaemia

Microspheres

Radioactive, fluorometric or colourmetric microspheres are injected centrally (left atrium, left ventricle or aorta). Regional blood flow is proportional to the number of microspheres trapped within the regional tissue being investigated.

 Sensitive (considered as the ‘gold standard’)  Allows delineation of regional muscle perfusion including deeper compartments

 Invasive  Technically challenging  Ionizing radiation (radioactive microspheres)  Reference blood sampling can cause hypotension in smaller animals

Contrast-enhanced direct injection X-ray

Contrast is injected intra-arterially. Arterial blood flow is assessed using serial X-ray or an X-ray image intensifier over the area of interest.

 Sensitive  Reproducible  Longitudinal acquisition of data

   

Invasive Technically challenging Ionizing radiation Not readily available

MRI angiography

Flow dependent or flow independent (contrast-enhanced) imaging of blood flow using Magnetic Resonance Imaging

   

Non-invasive Reproducible 3D-redendering Allows longitudinal acquisition of data  No ionizing radiation  Contrast agent less toxic than iodinated-contrast

   

Not readily available High cost Limited spatial resolution Long scan times

Micro-CT

Small scale version of computed tomography which provides significantly enhanced resolution.

    

Non-invasive Reproducible Excellent temporal resolution 3D-redendering Allows longitudinal acquisition of data

 Not readily available  High cost  Ionizing radiation

Contrast-Enhanced Ultrasound Sonography (CEUS)

Microbubble contrast agents are injected intra-arterially. Ultrasound sonography causes oscillation of microbubbles producing echogenic differences between contrast and tissues.

   

Low cost Sensitive Real-time evaluation of blood flow Microbubbles can be conjugated with ligands of interest (targeted CEUS)

 Short half-life of contrast agent  Immune-mediated removal and sequestration of contrast agent

Histological analysis

Various immunohistochemial and molecular markers can be used (e.g. CD31, VEGF etc.) to quantify microvessel density and factors within muscle to study the effect of ischaemia and the attenuating potential of novel therapeutics.

 Allows assessment of molecular changes  Tissue samples can be preserved for future analysis  Allows 3D-assessment (vascular casting or confocal microscopy)

 Time consuming  Requires sacrificing animal  Inter-assay variability

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arterial injections of radio- or fluorescently-labelled microspheres [28,38,39] are considered the gold standard technique for estimating regional blood flow which is proportional to the number of microspheres trapped in the area of interest. This relies on complete mixing of spheres in the central circulation (e.g. after injection into the ascending aorta or heart), total extraction in first pass and retention of spheres within the tissue of interest. Although this is a relatively laborious procedure, it is sensitive, allows different regions of tissue to be separated and provides information on deep muscle perfusion that is otherwise not amenable to imaging. Using the microsphere method in small animals like mice can be technically challenging. In order to accurately measure absolute rather than relative flow requires an arterial blood sample to be taken, which given the small total blood volume in the mouse, can result in hypotension [40]. Anaesthetic agents and compounds used to prevent aggregation of the microspheres can also cause hypotension which in turn affects the distribution of injected microspheres and may result in erroneous measurements [40]. Alternative methods of assessing limb perfusion are largely restricted to in vivo assessment of the collateral circulation using contrast-enhanced direct injection X-ray and MRI angiography [41,42]. The former technique has the disadvantage of producing 2Dimensional as opposed to 3-Dimensional images. Dynamic contrast-enhanced MRI offers the added opportunity to simultaneously quantify muscle perfusion in the hind limb and allow correlation of collateralisation with perfusion at muscle level [43]. Micro-CT is a particularly promising modality that provides 3D images with excellent spatial resolution (typically 1e100 mm) to allow quantification of neovessels and, to some extent, blood flow. This technique is, however, limited by the time taken to prepare and scan the animal and the relatively limited amount of image analysis software currently available [44e46]. Vasospasm and animal movement caused by the injection of contrast can result in blurred images with X-ray/MRI and CT angiography, making it more difficult to identify vessels. Finally, contrast-enhanced ultrasound sonography (CEUS) is a low cost alternative which provides imaging capabilities similar to the above techniques. CEUS incorporates the use of microbubble contrast agents which are injected intravenously in combination with traditional ultrasound sonography. The high frequency ultrasound waves cause the microbubble gas core to oscillate resulting in significant difference in tissue and contrast echogenicity. Advantages of CEUS include low cost, high sensitivity and assessment of both muscle perfusion and real-time evaluation of blood flow rate [47,48]. Microbubbles can also be conjugated with ligands to enable binding of receptors/molecular markers of interest (targeted CEUS) [47]. These have included endothelial adhesion molecules, ICAM-1 and P-selectin, to identify inflammatory regions and aV-integrins which are expressed on newly formed blood vessels to assess angiogenesis [49e51]. The short half-life of the microbubbles due to removal by immune cells or sequestration in the liver/spleen represents the main disadvantage of this technique [52]. Histological and molecular analysis of tissue samples is an alternative endpoint but requires sacrifice of the animal and therefore allows assessment at a single time point only [6]. Capillary density and capillary: fibre ratio, for example, can be quantified by staining muscle sections for an endothelial marker, such as CD31 or lectins. This is a common method for assessing the extent of neovascularisation in the ischaemic limb but preparing and analysing sections is time consuming and can result in inter-assay variability. To ascertain the 3D architecture and distribution of vessels directly requires either vascular casting [53], or a Z-stack acquisition from confocal microscopy [54]. Advances in image analysis such as deconvolution algorithms linked with light microscopy may improve intravital microscopy studies.

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5. Characteristics related to the animal Variability between mice. There is considerable variation in the distribution of collateral arteries in the lower limbs of healthy humans and the response of this collateral circulation following obstruction to arterial flow as a consequence of atherosclerotic disease varies between individuals [55]. This is thought to be the result of environmental and genetic factors that are as yet poorly understood. Mice appear to exhibit similar inter- and intra-strain differences in collateralisation. The two most common strains of mouse used for the HLIM, BALB/c and C57BL/6, are highly inbred strains with minimal genetic heterogeneity among animals, which would suggest that experiments using the same strain are reliable and reproducible [56]. There is, however, some variability between mice within the same strain; for example BALB/c mice demonstrated considerable inter-animal variation when stratified with regard to functionality of their pre-existing collaterals 24 h after femoral artery ligation [57]. One possible cause of variation in the endpoint measures observed and comparisons between the results of different research groups may be genetic drift, which is known to occur in highly inbred strains of mice. The differences in collateral conductance and remodelling are even more striking when BALB/c and A/J mice are compared with C57BL/6 mice [58]. The collateral reserve and response to ischaemia is greatest in C57BL/6 followed by A/J and then BALB/c mice. The hind limb circulation of A/J has more native collaterals with smaller diameters than found in BALB/c mice [59], but the A/J mouse exhibits greater collateral remodelling after induction of ischaemia. Consequently recovery in the A/J strain is faster. The BALB/c mouse is therefore considered the most appropriate strain in which to model CLI as it demonstrates slower recovery after induction of hind limb ischaemia and is more susceptible to tissue necrosis and limb loss after arterial ligation [42]. The C57BL/6 strain has a more complete recovery of limb perfusion after induction of ischaemia, more extensive pre-existing collateral artery network, and a greater capacity to recruit collateral arteries [42,60]. BALB/c mice exhibit poorer collateral growth in response to femoral artery ligation, but show a greater increase in capillary density (angiogenesis) within the ischaemic hind limb muscle compared with C57BL/6 mice [60]. Interstrain differences in the angiogenic and inflammatory response has also been demonstrated by a recent study comparing three different mouse strains (Swiss, BALB/c and C57BL/6), using a polyetherpolyurethane sponge implanted subcutaneously [61]. Inflammation is thought to be an important stimulus for angiogenesis, but while macrophage accumulation and activation was highest in the Swiss strain, this did not translate to increased neovascularisation of the sponge. The BALB/c mice had the highest levels of VEGF in their implants, which would correlate with the potent angiogenic response reported in this strain by other investigators [60]. Taken together, these findings indicate that increased capillary density and a greater capacity for angiogenesis does not appear to resolve ischaemia within the limb suggesting that collateral growth through arteriogenesis may be the major contributor to increased tissue perfusion and limb salvage in the hind limb model. Angiogenesis produces relatively small, thin-walled capillaries that are less robust than the collateral network that forms by arteriogenesis and are less effective in revascularising the ischaemic hind limb [62]. On the basis of collateral reserve, BALB/c mice seem to be a more appropriate strain to use for simulating CLI, whereas C57BL/ 6 may be better suited as a model for intermittent claudication. Claudication is a milder sequela of PAD in man where a transient arterial insufficiency occurs in the limb only when oxygen demand is increased by walking and not at rest. Age. The majority of patients with PAD and CLI are older and their regenerative capacity and dynamic response to stimuli is

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accordingly depressed. A 3 year-old mouse would be the equivalent of an 80 year-old human making it more appropriate to use older mice to model CLI [63]. The husbandry costs may, however, make such experiments prohibitively expensive. Mice that are aged demonstrate slower recovery rates after femoral artery ligation compared with young mice, and are less likely to recover completely without therapeutic interventions. Young, adult (3 month-old) mice had a 50% better functional recovery compared with aged (18 month-old) mice 14 days after induction of hind limb ischaemia [64]. Arteriogenesis appears to be diminished in the ischaemic hind limb of older mice and therefore revascularisation occurs predominantly through angiogenesis, which is less effective in this model. Collaterals in aged mice are less able to remodel and enlarge in response to femoral artery ligation because of a deficiency in eNOS production and increased susceptibility of their endothelial and smooth muscle cells to apoptosis [65]. Recovery of limb perfusion in the ischaemic hind limb in aged mice is further hindered by a relative deficiency in HIF-1alpha that results in reduced production of angiogenic factors and recruitment of angiogenic cells [66]. The effects of ageing in the hind limb ischaemia model have been investigated using ‘klotho’ mutant (kl/ kl) mice that show typical age-related phenotypes, such as arteriosclerosis, skin atrophy and osteoporosis [67]. Neovascularisation and recovery of the ischaemic limb was impaired in these mice as a consequence of reduced angiogenesis associated with an inhibition of endothelium-derived nitric oxide release from existing vasculature and also reduced vasculogenesis evidenced by a reduction in the number of bone marrow derived cells that had incorporated into the neovasculature of the hind limb [67]. Co-morbidities. Young, wild-type mice do not have any of the comorbidities commonly seen in CLI patients, such as diabetes mellitus, hypertension and hypercholesterolaemia [68]. A common sequela of these conditions is induction of inflammation and oxidative stress, leading to endothelial dysfunction [69] and impaired arteriogenesis, in part through a decrease in both flow-mediated dilatation and outward vascular remodelling [70]. Diabetes appears to impair arteriogenesis by blunting the response to shear stress, decreasing monocyte activation, and inhibiting the mobilisation and integration of progenitor cells in the endothelium [71]. The db/db mouse, which expresses a defective leptin receptor, is the most commonly used mouse model of Type-2 diabetes mellitus [72]. These mice eat excessively and become obese, dyslipidaemic, insulin resistant and eventually diabetic. Recovery of the ischaemic hind limb after femoral artery ligation in these mice is attenuated due to smaller collateral vessels in the limb, a reduction in the number of bone marrow derived progenitors mobilised into the circulation and diminished progenitor cell function [73]. The data on bone marrow derived progenitors obtained using the mouse model mirrors findings in patients with diabetes [74,75]. Niacin has recently been shown to accelerate hind limb neovascularisation in diabetic mice by enhancing endothelial progenitor cell mobilisation and improving the function of mobilised cells [76]. Alternative reasons for the impairment of recovery of the ischaemic hind limb in diabetic mice include reduced expression of platelet-derived growth factor B-chain homodimer (PDGF-BB) and defective vascular endothelial growth factor (VEGF) induced angiogenic signalling [77,78]. ApoE-knockout (ApoE/) and LDL receptor knockout (LDLR/) mice are commonly used to simulate the effects of atherosclerosis and dyslipidaemia. ApoE/ C57BL/6 mice develop significant atherosclerotic lesions in the ascending aorta, carotid, femoral and poplitaeal arteries [79], exhibit delayed recovery from ischaemia and respond poorly to angiogenic therapy with bone marrow mononuclear cells [80,81]. Hypercholesterolaemic mice have reduced expression of FGF receptor 1 and hypoxia-inducible factor1a [82]. This is associated with an attenuated arteriogenic response

in the ischaemic hind limb which correlates with the delayed recruitment of F4/80 þ macrophages in the tissue. Aged, hypercholesterolaemic ApoE-/- mice have been proposed as an appropriate transgenic strain for assessing hind limb ischaemia whilst incorporating two characteristics that are common in patients with CLI [83], but atherosclerotic plaques that form in mice are less prone to thrombosis and rupture than their counterparts in man [56,84]. Transgenic mice are useful for modelling the effects of specific genetic anomalies, but cannot fully replicate the human condition, particularly chronic multi-factorial polygenic diseases such as atherosclerosis. The LDLR/ mouse, for example, is technically a model of homozygous familial hypercholesterolaemia which only occurs in 1 in 1 million live births, and is not representative of the hypercholesterolaemia that occurs much more frequently in the general population [56]. Patients with homozygous familial hypercholesterolaemia have an unusual phenotype in that they suffer lower rates of myocardial infarction and stroke compared with patients with non-familial hypercholesterolaemia. The data from studies that use the LDLR/ mouse should therefore be interpreted with caution [56]. Manipulating the diet of the mouse prior to induction of hind limb ischaemia may be a better method of simulating the co-morbidities encountered in CLI patients than using transgenic mice. Mice fed a diet consisting of high fat and sucrose for 9 weeks develop obesity, hyperglycaemia, hyperinsulinaemia and endothelial dysfunction [85]. ApoE-knockout mice fed on such a diet would allow assessment of therapeutic interventions in the hind limb ischaemia model whilst incorporating the effects of multiple co-morbidities. Incorporating multiple morbidities stimulates the vascular dysfunction seen in CLI patients occurs through the complex interaction of multiple disease processes. Diabetic, atherosclerotic mice (double-knockout ApoE/  db/db/) maintained on a regular chow diet develop accelerated atherosclerosis and may be another candidate for better simulating the human condition for the proportion of patients with CLI who are diabetic [86]. Both genetic and non-genetic mouse models of hypertension exist [87]. Non-genetic models of hypertension include surgical induction of hypertension (e.g. constriction of a renal artery), dietinduced (e.g. high-salt diet, obesity) and drug-induced (e.g. angiotensin II). Genetic models include cross-bred selected spontaneously hypertensive mice (e.g. BPH/2 mouse), transgenic mice and gene knockout mice. Genes used for hypertensive KO mice have included nitric oxide synthase, endothelin, components of the renin-angiotensin-aldosterone pathway and sympathetic nervous system pathway (dopamine, alpha- and beta-adrenergic receptors) and natriuretic peptides (ANP, BNP). The deleterious effects of hypertension and endothelial dysfunction are cumulative and can exist in a vicious cycle. Hypertension-induced endothelial dysfunction results in microvascular rarefaction through capillary and arteriolar regression which exacerbates hypertension through subsequent increase in total peripheral resistance [88]. Moreover, attenuated angiogenic potential in mice results in significant increases in mean arterial pressure via endothelial dysfunction [89]; a phenomenon mirrored in patients with malignancy whereby anti-angiogenic therapy (e.g. Bevacizumab) promotes hypertension [90]. Angiogenesis in response to hind limb ischaemia is significantly attenuated in hypertensive rodents due to endothelial dysfunction, reduced eNOS expression and defective Akt associated angiogenic signalling [91,92]. It has been demonstrated that attenuating endothelial dysfunction by inducing eNOS expression increases capillary density through angiogenesis and improves blood flow recovery in a murine HLIM [93]. Thus, an alternative candidate for simulating the effect of co-morbidities would be an ApoE-/- and eNOS-/- double-knockout mouse that develops extensive atherosclerotic lesions, is hypertensive and by

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virtue of eNOS deficiency incorporate some of the biological effects of ageing on collateralisation that has been discussed earlier [94]. 6. Conclusion The incidence of CLI is rising as risk factors in an increasingly elderly population become more prevalent. There is considerable demand for novel treatment modalities such as therapeutic neovascularisation and consequently each new discovery that appears to hold promise is greeted with much excitement. An imperative of the translational research approach is to remain objective about the potential of each treatment before use in man. The limitations of pre-clinical assays such as the hind limb ischaemia model must be recognised and addressed in an effort to discover treatments that are likely to be equally as effective at the bedside as they are at the bench. The operative technique, endpoint measures and characteristics of the mice used have not been standardised by investigators that work within this field and there is a danger that researchers from different groups may not be comparing like for like. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements Bijan Modarai is funded by a British Heart Foundation Intermediate Clinical Research Fellowship and by the Circulation Foundation. Ashish Patel is funded through Clinical Research Fellowships from the BRC (at KHP) and the British Heart Foundation. References [1] Attanasio S, Snell J. Therapeutic angiogenesis in the management of critical limb ischemia: current concepts and review. Cardiol Rev 2009;17(3):115e20 [Epub 2009/04/23]. [2] Aranguren XL, Verfaillie CM, Luttun A. Emerging hurdles in stem cell therapy for peripheral vascular disease. J Mol Med 2009;87(1):3e16 [Epub 2008/08/21]. [3] Emanueli C, Madeddu P. Therapeutic angiogenesis: translating experimental concepts to medically relevant goals. Vascul Pharmacol 2006;45(5):334e9 [Epub 2006/09/30]. [4] Heil M, Eitenmuller I, Schmitz-Rixen T, Schaper W. Arteriogenesis versus angiogenesis: similarities and differences. J Cell Mol Med 2006;10(1):45e55 [Epub 2006/03/28]. [5] Lawall H, Bramlage P, Amann B. Treatment of peripheral arterial disease using stem and progenitor cell therapy. J Vasc Surg 2011;53(2):445e53 [Epub 2010/ 10/30]. [6] Madeddu P, Emanueli C, Spillmann F, et al. Murine models of myocardial and limb ischemia: diagnostic end-points and relevance to clinical problems. Vascul Pharmacol 2006;45(5):281e301 [Epub 2006/10/03]. [7] Leeper NJ, Hunter AL, Cooke JP. Stem cell therapy for vascular regeneration: adult, embryonic, and induced pluripotent stem cells. Circulation 2010; 122(5):517e26 [Epub 2010/08/04]. [8] Staton CA, Reed MW, Brown NJ. A critical analysis of current in vitro and in vivo angiogenesis assays. Int J Exp Pathol 2009;90(3):195e221 [Epub 2009/07/01]. [9] Hudlicka O, Brown M, Egginton S. Angiogenesis in skeletal and cardiac muscle. Physiol Rev 1992;72(2):369e417 [Epub 1992/04/01]. [10] Hudlicka O, Brown MD, Egginton S, Dawson JM. Effect of long-term electrical stimulation on vascular supply and fatigue in chronically ischemic muscles. J Appl Physiol 1994;77(3):1317e24 [Epub 1994/09/01]. [11] Becit N, Ceviz M, Kocak H, et al. The effect of vascular endothelial growth factor on angiogenesis: an experimental study. Eur J Vasc Endovasc Surg: Off J Eur Soc Vasc Surg 2001;22(4):310e6 [Epub 2001/09/21]. [12] Yasumura EG, Stilhano RS, Samoto VY, et al. Treatment of mouse limb ischemia with an integrative hypoxia-responsive vector expressing the vascular endothelial growth factor gene. PloS One 2012;7(3):e33944 [Epub 2012/04/04]. [13] Masaki I, Yonemitsu Y, Yamashita A, et al. Angiogenic gene therapy for experimental critical limb ischemia: acceleration of limb loss by overexpression of vascular endothelial growth factor 165 but not of fibroblast growth factor-2. Circ Res 2002;90(9):966e73 [Epub 2002/05/23]. [14] Rissanen TT, Korpisalo P, Karvinen H, et al. High-resolution ultrasound perfusion imaging of therapeutic angiogenesis. JACC Cardiovasc Imag 2008; 1(1):83e91 [Epub 2008/01/01].

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