Peripheral arterial disease (PAD) usually results from atherosclerosis and associated thrombosis and limits blood supply to the lower limbs. Common presenting symptoms include intermittent claudication (IC), rest pain and tissue loss. When limb viability is threatened, known as critical limb ischaemia (CLI), surgical and endovascular interventions are frequently undertaken; however, these are not always successful and ultimately major amputation may be required. There is significant interest in developing new therapeutic approaches to manage PAD which can be applied to patients unlikely to benefit from interventional approaches. Many of the therapeutic agents successful in inducing angiogenesis and arteriogenesis in pre-clinical animal models of PAD have failed to have efficacy in human randomized control trials. One possible reason for this inability to translate findings to patients could be the type of pre-clinical animal models used. In the present review, we describe currently available pre-clinical models of PAD and discuss the advantages and disadvantages of the available models. A detailed assessment of the currently available pre-clinical animal models shows major limitations such as variability in the surgical procedure used to induce limb ischaemia, variability in the strains of rodents used, lack of risk factors incorporated into the model and lack of standardized functional outcomes. The most commonly used outcome assessments in studies within pre-clinical models differ from those employed in clinical trials within PAD patients. Most current pre-clinical models are designed to produce acute ischaemia which leads to muscle necrosis and inflammation. Patients, however, most commonly present with chronic ischaemia suggesting that more representative models are needed to evaluate therapeutic modalities that can be potentially translated to clinical practice.

INTRODUCTION

Peripheral arterial disease (PAD) is usually caused by atherosclerosis and associated thrombosis which leads to narrowing and blockage of the arteries supplying blood to the lower limbs [1]. The prevalence of PAD in people aged ≥ 55 years is estimated to be approximately 10% and increases to approximately 40% in individuals aged > 80 years [2,3]. PAD patients frequently present with a slow onset of chronic leg ischaemia associated with symptoms of intermittent claudication (IC), rest pain, arterial ulceration or gangrene. Other patients with PAD may present with atypical leg symptoms affecting limb function or be asymptomatic [4]. Rarely patients with PAD may present with acute limb ischaemia (ALI) which suddenly threatens limb viability due to thrombotic occlusion of a lower limb artery which has associated atherosclerosis [4]. PAD patients usually seek medical assistance in order to improve their health-related quality of life which is limited due to impaired mobility and because of concerns regarding limb viability, known as critical limb ischaemia (CLI). Patients with CLI have a high risk of limb loss, a frequent requirement for hospital admission and surgical interventions and a high rate of fatal or non-fatal cardiovascular events, such as myocardial infarction and stroke [57]. Interventions for limb preservation are frequently not durable and re-intervention and ultimate requirement for major amputation are not unusual [59].

There is need for new therapeutic approaches for patients with PAD. Pre-clinical animal models are potentially indispensable tools for the development of novel therapies, the assessment of medical interventions and the study of molecular pathways involved in disease development. Improved design and development of animal models are warranted for successful translation of agents from animal models to clinical practice. The present review highlights several important aspects of PAD pathology and mechanisms of revascularization. The review also includes potential ways to improve the translational value of current pre-clinical models for PAD.

PAD RISK FACTORS AND DISEASE MANAGEMENT

Important risk factors for PAD are older age, male sex, smoking, diabetes, hypertension, dyslipidaemia and obesity [1014]. Current clinical management of PAD focuses on modifying these risk factors by lifestyle modification and medications and consideration of endovascular and surgical interventions aimed at improving limb perfusion [11,15,16]. Up to 30% of patients are not considered suitable for interventional revascularization, mainly due to unfavourable vascular involvement, associated operative risk and previous failed interventions [17,18]. Conventional revascularization procedures are successful in improving clinical symptoms of PAD in many instances, however new methods of inducing revascularization are needed and are actively being investigated [19,20].

NOVEL APPROACHES TO THERAPEUTIC REVASCULARIZATION

The focus for a number of novel interventions is to promote arteriogenesis or angiogenesis which are two key mechanisms of compensatory vessel growth (Figure 1). Following occlusion of the major arteries to the limb [such as the femoral artery (FA)] pre-existing arteriolar connections are recruited to bypass the site of occlusion through the process called arteriogenesis. Arteriogenesis is the rapid transformation of pre-existing large collateral vessels, small arteries, arterioles and capillaries into functional collateral arteries (collateral growth). This is naturally stimulated to a variable extent in patients with PAD. Narrowing or occlusions of lower limb arteries promotes changes in shear stress in pre-existing small arteries and arterioles. Elevated shear stress promotes inflammation, recruitment of monocytes and release of various growth factors such as platelet-derived growth factors (PDGFs) and monocyte chemoattractant protein (MCP)-1, which promote remodelling of arteries. The remodelled arteries have enhanced pericyte coverage and increased diameter thereby compensating to some extent for the occluded arteries [21,22].

Simplified scheme of therapeutic revascularization targets for PAD

Figure 1
Simplified scheme of therapeutic revascularization targets for PAD

Lower limb ischaemia occurs due to atherosclerotic occlusion in the vessels supplying blood to the leg. Revascularization targets in peripheral artery disease include arteriogenesis and angiogenesis. Arteriogenesis describes the process by which pre-existing arteriolar connections are recruited to bypass the site of occlusion. Occlusions of lower limb arteries cause changes in shear stress in pre-existing small arteries and arterioles promoting inflammation, recruitment of monocytes and up-regulation of various proteins such as PDGF, MCP-1 and NOS that promotes remodelling of arteries. The remodelled arteries have enhanced pericyte coverage, increased diameter, endothelial lining, an internal elastic lamina and vascular smooth muscle cells (VSMCs) thereby compensating to some extent for the occluded arteries. In response to local hypoxia in the ischaemic limb, sprouting of small endothelial tubes occurs from pre-existing capillary beds. The sprouting of a new capillary network (capillary growth) called angiogenesis is mediated through the activation, proliferation and migration of ECs, extracellular proteolysis and vascular wall remodelling. A number of growth factors, such as HIF-1, VEGF, as well as cytokines play a role in this process.

Figure 1
Simplified scheme of therapeutic revascularization targets for PAD

Lower limb ischaemia occurs due to atherosclerotic occlusion in the vessels supplying blood to the leg. Revascularization targets in peripheral artery disease include arteriogenesis and angiogenesis. Arteriogenesis describes the process by which pre-existing arteriolar connections are recruited to bypass the site of occlusion. Occlusions of lower limb arteries cause changes in shear stress in pre-existing small arteries and arterioles promoting inflammation, recruitment of monocytes and up-regulation of various proteins such as PDGF, MCP-1 and NOS that promotes remodelling of arteries. The remodelled arteries have enhanced pericyte coverage, increased diameter, endothelial lining, an internal elastic lamina and vascular smooth muscle cells (VSMCs) thereby compensating to some extent for the occluded arteries. In response to local hypoxia in the ischaemic limb, sprouting of small endothelial tubes occurs from pre-existing capillary beds. The sprouting of a new capillary network (capillary growth) called angiogenesis is mediated through the activation, proliferation and migration of ECs, extracellular proteolysis and vascular wall remodelling. A number of growth factors, such as HIF-1, VEGF, as well as cytokines play a role in this process.

In response to local hypoxia in the ischaemic limb, sprouting of small endothelial tubes occurs from pre-existing capillary beds. The sprouting of a new capillary network (capillary growth) is mediated through the activation, proliferation and migration of endothelial cells (ECs), extracellular proteolysis and vascular wall remodelling. This process is called angiogenesis. A number of growth factors, such as hypoxia-inducible growth factors (HIF-1) and vascular endothelial growth factor (VEGF), as well as cytokines play a role in this process. This eventually results in the formation of new capillaries [21].

Therapeutic revascularization procedures focused on promoting arteriogenesis and/ or angiogenesis have potential to ultimately assist patients with PAD although this is yet to be fully appreciated in clinical practice (Figure 1).

NOVEL THERAPIES ASSESSED IN ANIMAL MODELS OF PAD

A number of novel therapies for PAD have been tested in pre-clinical models over the last decade (see examples in Table 1). Animal studies suggest that a number of recombinant proteins, mitogenic agents and growth factors stimulate the development of new blood vessels in the models tested [23,24]. Intra-arterial administration of recombinant fibroblast growth factor (FGF) was reported to be beneficial in patients with PAD in the Therapeutic Angiogenesis with FGF-2 for intermittent claudication (TRAFFIC) study [25]. However, the phase III Therapeutic Angiogenesis for the Management of Arteriopathy in a Randomised International Study (TAMARIS) trial (involving patients from 30 different countries) suggested that non-viral-FGF gene therapy was not effective in reducing major amputation or death in PAD patients. Similarly, initial studies using VEGF have not been positive. The double-blinded randomized placebo-controlled phase II study named Regional Angiogenesis with VEGF in PAD (RAVE) showed no improvement in the primary efficacy end-point which was improvement in walking time [26]. A double-blinded placebo-controlled study assessing the effect of plasmid-encoded angiomatrix protein named development-regulated endothelial locus-1 (Del-1) for the treatment of IC [Del-1 for Therapeutic Angiogenesis Trial (DELTA-1)] also did not find any benefit in the main outcomes assessed such as peak walking time [27]. A number of interventions, such as non-viral 1 FGF transfer (NV1FGF), which have been successful in animal models, have not proved to be effective in trials in PAD patients [28,29].

Table 1
Examples of interventions tested in pre-clinical animal models.

Abbreviations: AdV, adeno virus; CD, cluster of differentiation; HGF, hepatocyte growth factor; IL, interleukin; IM, intramuscular; NOD, non-obese diabetic; MMP, matrix metalloproteinase; NZW, New Zealand White rabbits; SCID, severe combined immunodeficiency; SDF, stromal-derived factor.

 Therapies tested Animal used for HLI Mode of therapy and sample size Observations 
Gene therapy AdV-mediated VEGF; VEGF121 and VEGF165 NZW rabbits IM injection (n=35) VEGF overexpression promotes growth of arteries and veins [38
 AdV-mediated VEGF-A and PDGF-B  IM injection (n=58) Increased angiogenesis, increased perfusion, no change in pericyte recruitment [39
 AdV-mediated VEGF-A and VEGF-D gene transfer  IM injection (n=66) Increased microvessel enlargement and pericyte recruitment to form arterioles or venules [40
 AdV-mediated VEGF-A  Retroinfusion into the anterior tibial vein (n=5/group) Increased collateral growth [41
 AdV-mediated VEGF and AdV-mediated FGF-4  IM injection (n=7/group) Increased vascular permeability and increased collateral growth, popliteal blood flow and muscle perfusion [42
 AdV-mediated HIF-1α  IM injection (n=8) Improved tissue perfusion and collateral vessels [43
 AdV encoding a constitutively active form of HIF-1  IM injection (n=18) Recovery of limb perfusion through increased arterial remodelling, arteriogenesis and angiogenesis [44
 Naked DNA hybrid expressing two isoforms of HGF  IM injection (n=16) Increased blood flow and formation of collateral artery [45
 AdV-mediated placental growth factor131  IM injection (n=10/group) Improved performance of ischaemic limbs [46
 Plasmid or cDNA hybrids with human HGF gene  Intra-arterial injection (n=7–8/group) Perfusion recovery, reduction in severe necrosis, no significant change in haematology or serum chemistry [47
 Plasmid encoding human VEGF165  IM injection (n=10/group) Increased microvasculature and reduced ischaemic muscle damage [48
   Percutaneous delivery using an angioplasty balloon (n=5/group) Development of collateral vessels [34]. 
 Vector-based siRNA-mediated gene knockdown Sprague–Dawley rats IM injection (n=6/group) Increased capillary density [50
 AdV-mediated gene transfer of VEGF121 Sprague–Dawley rats and NZW rabbits IM injection (n=64) Increased tissue perfusion and collateral vessels [51
 Plasmid vector with the hypoxia-induced phiC31 integrative system for long-term VEGF gene expression BALB/c mice IM injection (n=7–10/group) High concentration of VEGF in ischaemic tissue results in muscle necrosis [52
 Recombinant Sendai virus (SeV) mediated overexpression of FGF-2 and VEGF165 C57BL/6 mice and BALB/c nu/nu mice IM injection (n=10/group) VEGF and FGF-2 overexpression results in limb amputation without recovery of blood perfusion; these agents worsened results [53
 Adenovirus-mediated human tissue Kallikrein Streptozotocin-induced type-1 diabetic mice IM injection (n=10/group) Improved perfusion recovery; improved neovascularization, suppressed apoptosis and up-regulated eNOS expression [54
Recombinant protein therapy SDF-1 resistant to MMP cleavage C57BL/6 mice IM injection (n=10/group) Increased blood flow and formation of new arterioles [40
 Human IL-11 mediated CD34(+)/VEGF-receptor-2(+) mononuclear cell mobilization Sv129 mice Subcutaneous mini-pumps (n=9) Increased blood flow, increase in collateral vessel luminal diameter, better hindlimb appearance and use score [55
 Intra-arterially administered VEGF NZW rabbits IM injection (n=7/group) Improved thigh pressure index and formation of collaterals [56
 Effect of natural compound propionyl-L-carnitine on ischaemic tissue  IM injection (n=9) Increased blood flow and revascularization, reduced endothelial NADPH-oxidase-related superoxide production [57
Cell-based therapy Human cord blood-derived mononuclear cells Dogs IM injection (n=12) Increase in capillary numbers and expression of genes associated with angiogenesis [30
 Bone marrow-derived MSCs encapsulated in a radiopaque contrast agent (MSC-Xcaps) NZW rabbits IM injection (n=21) Improvement in hind limb perfusion and arteriogenesis [35
 Co-treatment with autologous bone marrow cells, vitamin E, vitamin C and L-arginine Diabetic CD1 mice Intravenous injection (n=18/group) Increased blood flow, capillary density and decreased interstitial fibrosis [31
 Effect of T-cell-pre-stimulated human monocytes Nude C57BL/6 mice and NOD-SCID-IL2-Rγ mice IM injection (n=10/group) Improved blood flow recovery, increased size and number of collaterals and capillaries [32
 ECs derived from hiPSCs NOD SCID mice IM injection (n=8/group) Increased perfusion and number of capillaries [58
 Adipose tissue-derived regenerative cells from Lewis rats BALB/c-nu/nu mice IM injection (n=8/group) Improved blood perfusion, capillary density and production of angiogenesis promoting factors [37
 Therapies tested Animal used for HLI Mode of therapy and sample size Observations 
Gene therapy AdV-mediated VEGF; VEGF121 and VEGF165 NZW rabbits IM injection (n=35) VEGF overexpression promotes growth of arteries and veins [38
 AdV-mediated VEGF-A and PDGF-B  IM injection (n=58) Increased angiogenesis, increased perfusion, no change in pericyte recruitment [39
 AdV-mediated VEGF-A and VEGF-D gene transfer  IM injection (n=66) Increased microvessel enlargement and pericyte recruitment to form arterioles or venules [40
 AdV-mediated VEGF-A  Retroinfusion into the anterior tibial vein (n=5/group) Increased collateral growth [41
 AdV-mediated VEGF and AdV-mediated FGF-4  IM injection (n=7/group) Increased vascular permeability and increased collateral growth, popliteal blood flow and muscle perfusion [42
 AdV-mediated HIF-1α  IM injection (n=8) Improved tissue perfusion and collateral vessels [43
 AdV encoding a constitutively active form of HIF-1  IM injection (n=18) Recovery of limb perfusion through increased arterial remodelling, arteriogenesis and angiogenesis [44
 Naked DNA hybrid expressing two isoforms of HGF  IM injection (n=16) Increased blood flow and formation of collateral artery [45
 AdV-mediated placental growth factor131  IM injection (n=10/group) Improved performance of ischaemic limbs [46
 Plasmid or cDNA hybrids with human HGF gene  Intra-arterial injection (n=7–8/group) Perfusion recovery, reduction in severe necrosis, no significant change in haematology or serum chemistry [47
 Plasmid encoding human VEGF165  IM injection (n=10/group) Increased microvasculature and reduced ischaemic muscle damage [48
   Percutaneous delivery using an angioplasty balloon (n=5/group) Development of collateral vessels [34]. 
 Vector-based siRNA-mediated gene knockdown Sprague–Dawley rats IM injection (n=6/group) Increased capillary density [50
 AdV-mediated gene transfer of VEGF121 Sprague–Dawley rats and NZW rabbits IM injection (n=64) Increased tissue perfusion and collateral vessels [51
 Plasmid vector with the hypoxia-induced phiC31 integrative system for long-term VEGF gene expression BALB/c mice IM injection (n=7–10/group) High concentration of VEGF in ischaemic tissue results in muscle necrosis [52
 Recombinant Sendai virus (SeV) mediated overexpression of FGF-2 and VEGF165 C57BL/6 mice and BALB/c nu/nu mice IM injection (n=10/group) VEGF and FGF-2 overexpression results in limb amputation without recovery of blood perfusion; these agents worsened results [53
 Adenovirus-mediated human tissue Kallikrein Streptozotocin-induced type-1 diabetic mice IM injection (n=10/group) Improved perfusion recovery; improved neovascularization, suppressed apoptosis and up-regulated eNOS expression [54
Recombinant protein therapy SDF-1 resistant to MMP cleavage C57BL/6 mice IM injection (n=10/group) Increased blood flow and formation of new arterioles [40
 Human IL-11 mediated CD34(+)/VEGF-receptor-2(+) mononuclear cell mobilization Sv129 mice Subcutaneous mini-pumps (n=9) Increased blood flow, increase in collateral vessel luminal diameter, better hindlimb appearance and use score [55
 Intra-arterially administered VEGF NZW rabbits IM injection (n=7/group) Improved thigh pressure index and formation of collaterals [56
 Effect of natural compound propionyl-L-carnitine on ischaemic tissue  IM injection (n=9) Increased blood flow and revascularization, reduced endothelial NADPH-oxidase-related superoxide production [57
Cell-based therapy Human cord blood-derived mononuclear cells Dogs IM injection (n=12) Increase in capillary numbers and expression of genes associated with angiogenesis [30
 Bone marrow-derived MSCs encapsulated in a radiopaque contrast agent (MSC-Xcaps) NZW rabbits IM injection (n=21) Improvement in hind limb perfusion and arteriogenesis [35
 Co-treatment with autologous bone marrow cells, vitamin E, vitamin C and L-arginine Diabetic CD1 mice Intravenous injection (n=18/group) Increased blood flow, capillary density and decreased interstitial fibrosis [31
 Effect of T-cell-pre-stimulated human monocytes Nude C57BL/6 mice and NOD-SCID-IL2-Rγ mice IM injection (n=10/group) Improved blood flow recovery, increased size and number of collaterals and capillaries [32
 ECs derived from hiPSCs NOD SCID mice IM injection (n=8/group) Increased perfusion and number of capillaries [58
 Adipose tissue-derived regenerative cells from Lewis rats BALB/c-nu/nu mice IM injection (n=8/group) Improved blood perfusion, capillary density and production of angiogenesis promoting factors [37

Cell-based regenerative therapy currently appears a more promising therapy for PAD. Some of the cell-based regenerative therapies tested in animal models include transplantation of human cord blood-derived mononuclear cells, transfusion of T-cell-pre-stimulated monocytes and autologous bone marrow cell therapy [30,31,32]. Stem cell therapies using adult or embryonic stem cells and ECs derived from human-induced pluripotent stem cells (hiPSCs) [58], endothelial progenitor cells (EPCs) [33,34], bone marrow-derived mesenchymal stem cells (MSCs) [35], muscle-derived stem cells (MDSCs) [36] and adipose-derived regenerative cells (cADRCs) [37], have also been shown to induce angiogenesis and promote revascularization in animal models of hind limb ischaemia (HLI) (Table 2).

Table 2
Examples of cellular and molecular interventions tested in unilateral HLI animal models and clinical trials

Only the pre-clinical studies referenced by the clinical trial paper for the respective therapy are mentioned.

Abbreviations: AdCMV, adenovirus cytomegalovirus vector; ATMSC, adipose tissue-derived MSCs; BM-MNC, bone marrow-derived mononuclear cells; CBP, calf blood pressure ratio; CCR, CC chemokine receptor; CD, cluster of differentiation; CIA, common iliac artery; DFA, deep FA; hADSC, human adipose-derived stem cells; HGF, hepatocyte growth factor; IHC, immunohistochemistry; IM, intramuscular; 99mTC, technetium; 7ND, 7 dominant negative; NZW, New Zealand white; PWT, peak walking time; RT, recombinant protein therapy; SA, saphenous artery; SeV, Sendai virus; SVF, stromal vascular fraction; TBPI, transcutaneous blood pressure index; VEC; vascular EC; WBS, Wong-Baker FACES Pain Rating Scale.

 Pre-clinical Clinical 
Cell-type tested Unilateral HLI model used Animal used for HLI induction Sample sizes/Groups Outcome assessed in animal models Results of animal studies/end-points assessed Outcomes of clinical trials 
BM-MNCs Ligation and excision of FA [70NZW rabbits Saline control (n=8), autologous BM-MNC (n=13), autologous BM-fibroblasts (n=6) LDPI, angiography, CBP ratio and IHC Increased blood perfusion, higher collateral vessels and capillary density, 4 weeks after transplantation in the BM-MNC transplanted group Treatment with autologous BM-MNCs resulted in marked clinical benefit in patients with severe symptomatic PAD with no further options for surgical and/or endovascular revascularization (n=16, Rutherford category 3–6) [61
ATMSCs Ligation of proximal FA [71BALB/c nude mice PBS control (n=10), hADSC (n=10) LDPI, histology and IHC Increased blood perfusion, increased vascular density and recovered muscle injury in the hADSC-treated group A safety and efficacy study in CLI male patients (n=15) showed that multiple IM injections with ATMSC improved clinical outcomes in 66.7% of patients which included the WBS, claudication walking distances and healing of ulcers [62
 Ligation and excision of FA and femoral vein [72BALB/c nude mice Endothelial medium control (n=11), unsorted hADSC (n=12) Perfusion imaging using a near IR perfusion imaging system and IHC Increased blood perfusion, increased vascular density, reduced muscle atrophy and reduced incidence of auto-amputation in hADSC treated group  
 Ligation and excision of FA [73C57BL/6J mice PBS control (n=8), mADRC (n=8), mature adipocyte (n=8) LDPI and IHC Increased blood perfusion, increased circulating EPCs and capillary density in the ischaemic skeletal site in the mADRC group  
 Ligation and excision of FA and SA [74C57BL/6J mice PBS control (n=10), BM cell (n=10), mature adipocyte control (n=4), SVF (n=10) LDPI and IHC Increased blood perfusion, increased vascular densities in BM cell and SVF-treated groups  
EPCs Ligation and excision of FA [75NZW rabbits Autologous MBCD34+ cell injection (n=4) Capillary density by IHC EPC localization was seen in capillaries and neovascular zones of the ischaemic limb Phase I/II clinical trial with IM injection of EPCs showed improvement in the WBS, TBPI, transcutaneous partial oxygen pressure, total or pain-free walking distance and ulcer size in patients (n=17) [63
 Ligation and excision of FA [76Athymic nude mice Control culture media (n=14), control hVEC (n=12), hEPC (n=17) LDPI & IHC Increased blood perfusion, increased capillary density and significantly reduced rate of limb loss in hEPC-treated animals Phase I clinical trial showed improvement in amputation free survival, pain relief, exercise capacity, collateral formation, perfusion and quality of life in CLI patients (n=9) receiving EPCs [64
HGF Ligation and excision of FA [47,77NZW rabbits Control (n=7–8), 100 μg of hHGF vector transfection (n=7–8), 250 μg of hHGF vector transfection (n=7–8), 500 μg of hHGF vector transfected HGF group (n=7–8) Quantitative angiography, intra-arterial guided wire measurement of blood flow and CBP ratio Increased blood perfusion, CBP ratio and collateral vessel development from the origin stem artery to the distal point of the reconstituted parent vessel was observed in rabbits receiving IM injection of hHGF plasmid once, 10 days after surgery Phase I/II study showed improved limb perfusion with HGF plasmid-treated CLI patients (n=78) compared with placebo-treated CLI patients (n=26). No differences were observed in the toe-brachial index, ABI, pain relief, wound healing or major amputation [65
   Sprague–Dawley rats Control (n=5–8), 100 μg of hHGF vector transfection (n=5–8), 250 μg of hHGF vector transfection (n=5–8) and 500 μg of hHGF vector transfected (n=5–8) LDPI and capillary density by IHC with alkaline phosphatase staining of ECs A dose-dependent increased blood perfusion was observed in both HGF and hHGF transfection Phase III study showed improvement in ulcer healing and quality of life, with no improvement in ischaemic rest pain and ulcer healing in CLI patients (n=27) treated with naked plasmid encoding hHGF gene compared with placebo (n=13) [78
FGF (1 and 2) Ligation and excision of FA and femoral vein [53C57BL/6 and BALB/c nu/nu mice PBS control (n=10), SeV luciferase control (n=10), SeV–FGF2 (n=10) and SeV–VEGF165 (n=10) LDPI, limb salvage score and IHC Increased blood perfusion, increased capillary densities and limb salvage with IM injection of SeV–FGF-2 compared with control vector, PBS and SeV–VEGF165; accelerated amputation, massive muscular oedema, necrosis and disturbed regeneration with IM injection of VEGF165; increased number of capillaries with no improvement in blood perfusion. Phase I/IIa clinical trial showed that DVC1-0101, a new vector-based human FGF-2 gene transfer (rSeV/dF–hFGF2), in PAD patients (n=12) with rest pain was safe and well tolerated and resulted in improvement of limb function [79
 Ligation and excision of FA and femoral vein and their branches up to and including the SA and PA bifurcation [80CCR deficient and BALB/c nu/nu mice Luciferase control (n=10), SeV luciferase (n=30), SeV mFGF-2 (30), mFGF-2 luciferase (n=10), mFGF-2 7ND MCP-1 (n=10) and luciferase 7ND MCP-1 (n=10) LDPI and IHC Dominant negative mutant-MCP-1 gene transfer diminished both adaptive and FGF-2-mediated recovery of blood flow; CCR2-deficient mice lost approximately 40% of their limbs compared with controls; SeV-mediated FGF2 gene transfer significantly but partially restored the limb survival of CCR2-deficient mice Phase II trial (TRAFFIC) in recombinant FGF-2 treated PAD patients (n=127) resulted in improved PWT and ABI compared with placebo-treated group (n=63) [25
 Ligation and excision of FA [81NZW rabbits pGSVLacZ control (n=10) non-secreted aFGF (p267, n=10), secreted aFGF (pMJ35, n=10) CBP ratio, internal iliac arteriography, capillary density and regional blood flow using coloured microspheres pMJ35 transfectants had increased angiographically visible collaterals, CBP ratio and capillary density and lower vascular resistance than either p267 or LacZ controls  
VEGF Ligation and excision of FA [82,83NZW rabbits Saline control (n=11) and EC growth factor (n=11) CBP ratio, 99mTC macro-aggregate calf radioisotopic perfusion scan, angiography Increased blood perfusion, CBP ratio and increased revascularization in growth factor-treated animals Phase II clinical trial (RAVE) showed that treatment with AdVEGF121 in PAD patients (n=72) did not improve PWT, quality of life and claudication onset time [26
 Ligation of CIA [84Sprague–Dawley rats PBS control (n=8), naive control (n=8), AdVEGF treated (n=8) and AdNull (n=8) Blood flow imaging by colour microsphere and 99mTC-labelled sestamib radionucleotide, angiography and histology Increased blood perfusion and greater vascularity in the Ad-VEGF-treated group Phase II clinical trial showed improvements in haemodynamics and skin ulcers, decreased pain, but no reduction in amputation in diabetic CLI patients treated with plasmid carrying VEGF165 gene (n=27) compared with placebo (n=27) [66
 Ligation and excision of FA [51,85Wistar rats PBS control (n=3), AdCMV–VEGF121 (n=6) and AdCMV.null (n=5) Bioenergetic reserve by NMR spectroscopy and IHC AdCMV–VEGF121-treated animals showed markedly improved bioenergetic reserve, capillary density, tissue perfusion and spontaneous collateral vessel development  
 Ligation and excision of FA [86Beagle dogs Saline control (n=6) and VEGF121 plasmid treated (n=6) CBP ratio, angiography, vasomotor reserve and haematological assays Increased collateral artery development, CBP ratio and blood supply in pVEGF121-treated groups. No significant modification in haematological variables Phase II trial showed increased vascularity in Ad.VEGF165 plasmid-treated groups (n=35) compared with placebo-treated group (n=19) [84
Del-1 Ligation and excision of FA [87NZW rabbits Non-coding plasmids control (n=3), hVEGF165 plasmid (n=6) and hDel-1 plasmid (n=4) Angiography, gene expression by RT-PCR and CD31 expression by Western blotting hVEGF and hDel-1 plasmid induced the formation of 3-fold more new blood vessels Phase II clinical trial showed improvements in PWT, claudication onset time, ABI in Del-1 plasmid-treated IC patients (n=52) and placebo-treated IC patients (n=53) 
 Ligation of FA [87CD1 mice Non-coding plasmid control (n=7–8), hVEGF165 plasmid (n=7–8) and hDel-1 plasmid (n=7–8) Treadmill run time, capillary myofibre ratio determination by IHC with CD31, Del-1 and VEGF165 hVEGF and Del-1 were equally effective in inducing neovessel formation and restoring hind-limb function There were no significant differences between groups [27
 Pre-clinical Clinical 
Cell-type tested Unilateral HLI model used Animal used for HLI induction Sample sizes/Groups Outcome assessed in animal models Results of animal studies/end-points assessed Outcomes of clinical trials 
BM-MNCs Ligation and excision of FA [70NZW rabbits Saline control (n=8), autologous BM-MNC (n=13), autologous BM-fibroblasts (n=6) LDPI, angiography, CBP ratio and IHC Increased blood perfusion, higher collateral vessels and capillary density, 4 weeks after transplantation in the BM-MNC transplanted group Treatment with autologous BM-MNCs resulted in marked clinical benefit in patients with severe symptomatic PAD with no further options for surgical and/or endovascular revascularization (n=16, Rutherford category 3–6) [61
ATMSCs Ligation of proximal FA [71BALB/c nude mice PBS control (n=10), hADSC (n=10) LDPI, histology and IHC Increased blood perfusion, increased vascular density and recovered muscle injury in the hADSC-treated group A safety and efficacy study in CLI male patients (n=15) showed that multiple IM injections with ATMSC improved clinical outcomes in 66.7% of patients which included the WBS, claudication walking distances and healing of ulcers [62
 Ligation and excision of FA and femoral vein [72BALB/c nude mice Endothelial medium control (n=11), unsorted hADSC (n=12) Perfusion imaging using a near IR perfusion imaging system and IHC Increased blood perfusion, increased vascular density, reduced muscle atrophy and reduced incidence of auto-amputation in hADSC treated group  
 Ligation and excision of FA [73C57BL/6J mice PBS control (n=8), mADRC (n=8), mature adipocyte (n=8) LDPI and IHC Increased blood perfusion, increased circulating EPCs and capillary density in the ischaemic skeletal site in the mADRC group  
 Ligation and excision of FA and SA [74C57BL/6J mice PBS control (n=10), BM cell (n=10), mature adipocyte control (n=4), SVF (n=10) LDPI and IHC Increased blood perfusion, increased vascular densities in BM cell and SVF-treated groups  
EPCs Ligation and excision of FA [75NZW rabbits Autologous MBCD34+ cell injection (n=4) Capillary density by IHC EPC localization was seen in capillaries and neovascular zones of the ischaemic limb Phase I/II clinical trial with IM injection of EPCs showed improvement in the WBS, TBPI, transcutaneous partial oxygen pressure, total or pain-free walking distance and ulcer size in patients (n=17) [63
 Ligation and excision of FA [76Athymic nude mice Control culture media (n=14), control hVEC (n=12), hEPC (n=17) LDPI & IHC Increased blood perfusion, increased capillary density and significantly reduced rate of limb loss in hEPC-treated animals Phase I clinical trial showed improvement in amputation free survival, pain relief, exercise capacity, collateral formation, perfusion and quality of life in CLI patients (n=9) receiving EPCs [64
HGF Ligation and excision of FA [47,77NZW rabbits Control (n=7–8), 100 μg of hHGF vector transfection (n=7–8), 250 μg of hHGF vector transfection (n=7–8), 500 μg of hHGF vector transfected HGF group (n=7–8) Quantitative angiography, intra-arterial guided wire measurement of blood flow and CBP ratio Increased blood perfusion, CBP ratio and collateral vessel development from the origin stem artery to the distal point of the reconstituted parent vessel was observed in rabbits receiving IM injection of hHGF plasmid once, 10 days after surgery Phase I/II study showed improved limb perfusion with HGF plasmid-treated CLI patients (n=78) compared with placebo-treated CLI patients (n=26). No differences were observed in the toe-brachial index, ABI, pain relief, wound healing or major amputation [65
   Sprague–Dawley rats Control (n=5–8), 100 μg of hHGF vector transfection (n=5–8), 250 μg of hHGF vector transfection (n=5–8) and 500 μg of hHGF vector transfected (n=5–8) LDPI and capillary density by IHC with alkaline phosphatase staining of ECs A dose-dependent increased blood perfusion was observed in both HGF and hHGF transfection Phase III study showed improvement in ulcer healing and quality of life, with no improvement in ischaemic rest pain and ulcer healing in CLI patients (n=27) treated with naked plasmid encoding hHGF gene compared with placebo (n=13) [78
FGF (1 and 2) Ligation and excision of FA and femoral vein [53C57BL/6 and BALB/c nu/nu mice PBS control (n=10), SeV luciferase control (n=10), SeV–FGF2 (n=10) and SeV–VEGF165 (n=10) LDPI, limb salvage score and IHC Increased blood perfusion, increased capillary densities and limb salvage with IM injection of SeV–FGF-2 compared with control vector, PBS and SeV–VEGF165; accelerated amputation, massive muscular oedema, necrosis and disturbed regeneration with IM injection of VEGF165; increased number of capillaries with no improvement in blood perfusion. Phase I/IIa clinical trial showed that DVC1-0101, a new vector-based human FGF-2 gene transfer (rSeV/dF–hFGF2), in PAD patients (n=12) with rest pain was safe and well tolerated and resulted in improvement of limb function [79
 Ligation and excision of FA and femoral vein and their branches up to and including the SA and PA bifurcation [80CCR deficient and BALB/c nu/nu mice Luciferase control (n=10), SeV luciferase (n=30), SeV mFGF-2 (30), mFGF-2 luciferase (n=10), mFGF-2 7ND MCP-1 (n=10) and luciferase 7ND MCP-1 (n=10) LDPI and IHC Dominant negative mutant-MCP-1 gene transfer diminished both adaptive and FGF-2-mediated recovery of blood flow; CCR2-deficient mice lost approximately 40% of their limbs compared with controls; SeV-mediated FGF2 gene transfer significantly but partially restored the limb survival of CCR2-deficient mice Phase II trial (TRAFFIC) in recombinant FGF-2 treated PAD patients (n=127) resulted in improved PWT and ABI compared with placebo-treated group (n=63) [25
 Ligation and excision of FA [81NZW rabbits pGSVLacZ control (n=10) non-secreted aFGF (p267, n=10), secreted aFGF (pMJ35, n=10) CBP ratio, internal iliac arteriography, capillary density and regional blood flow using coloured microspheres pMJ35 transfectants had increased angiographically visible collaterals, CBP ratio and capillary density and lower vascular resistance than either p267 or LacZ controls  
VEGF Ligation and excision of FA [82,83NZW rabbits Saline control (n=11) and EC growth factor (n=11) CBP ratio, 99mTC macro-aggregate calf radioisotopic perfusion scan, angiography Increased blood perfusion, CBP ratio and increased revascularization in growth factor-treated animals Phase II clinical trial (RAVE) showed that treatment with AdVEGF121 in PAD patients (n=72) did not improve PWT, quality of life and claudication onset time [26
 Ligation of CIA [84Sprague–Dawley rats PBS control (n=8), naive control (n=8), AdVEGF treated (n=8) and AdNull (n=8) Blood flow imaging by colour microsphere and 99mTC-labelled sestamib radionucleotide, angiography and histology Increased blood perfusion and greater vascularity in the Ad-VEGF-treated group Phase II clinical trial showed improvements in haemodynamics and skin ulcers, decreased pain, but no reduction in amputation in diabetic CLI patients treated with plasmid carrying VEGF165 gene (n=27) compared with placebo (n=27) [66
 Ligation and excision of FA [51,85Wistar rats PBS control (n=3), AdCMV–VEGF121 (n=6) and AdCMV.null (n=5) Bioenergetic reserve by NMR spectroscopy and IHC AdCMV–VEGF121-treated animals showed markedly improved bioenergetic reserve, capillary density, tissue perfusion and spontaneous collateral vessel development  
 Ligation and excision of FA [86Beagle dogs Saline control (n=6) and VEGF121 plasmid treated (n=6) CBP ratio, angiography, vasomotor reserve and haematological assays Increased collateral artery development, CBP ratio and blood supply in pVEGF121-treated groups. No significant modification in haematological variables Phase II trial showed increased vascularity in Ad.VEGF165 plasmid-treated groups (n=35) compared with placebo-treated group (n=19) [84
Del-1 Ligation and excision of FA [87NZW rabbits Non-coding plasmids control (n=3), hVEGF165 plasmid (n=6) and hDel-1 plasmid (n=4) Angiography, gene expression by RT-PCR and CD31 expression by Western blotting hVEGF and hDel-1 plasmid induced the formation of 3-fold more new blood vessels Phase II clinical trial showed improvements in PWT, claudication onset time, ABI in Del-1 plasmid-treated IC patients (n=52) and placebo-treated IC patients (n=53) 
 Ligation of FA [87CD1 mice Non-coding plasmid control (n=7–8), hVEGF165 plasmid (n=7–8) and hDel-1 plasmid (n=7–8) Treadmill run time, capillary myofibre ratio determination by IHC with CD31, Del-1 and VEGF165 hVEGF and Del-1 were equally effective in inducing neovessel formation and restoring hind-limb function There were no significant differences between groups [27

LIMITED CURRENT ABILITY TO TRANSLATE PROMISING PRE-CLINICAL FINDINGS

Findings from current pre-clinical models of PAD have been hard to translate to patients as illustrated in Table 2 [17,59,60]. Cell therapies shown to be successful in animal models have shown promising results in terms of safety and efficacy in trials involving small numbers of PAD patients [61,62,63,64]. Whether these results can be confirmed in large randomized trials however remains to be seen. Gene therapies which were highly successful in pre-clinical studies have been largely unsuccessful in phase II and III clinical trials with limited improvement in ischaemic pain, wound healing and no change in the rate of major amputation [26,65,66] (Table 2). Thus, even though some of the cell-based therapies look promising, it is important that these small-scale clinical trials are followed up by large studies with sufficient follow-up to examine whether the therapy is safe and effective. A number of reviews and meta-analyses exist in this area to which the readers are referred [6769].

There are probably a number of reasons for the inability to translate finding from pre-clinical studies to humans, such as poor study design, absence of validation studies, insufficient sample sizes and inappropriate models. In order for findings from pre-clinical animal models to be relevant to patients, it would seem important that the animal model recapitulates the disease mechanisms in humans. The failure of some agents tested in humans which were found to be effective in animal models prompted us to evaluate the currently available animal models of PAD in the present review.

CURRENT PRE-CLINICAL PAD MODELS

Ideally, an animal model for PAD should closely resemble the pathological and functional characteristics of the common human presentations, such as IC and CLI. Other important characteristics of an ideal animal model for PAD include: (i) the model would have similar responses to the known determinants of the human disease, i.e. the determinants of outcome within the model would be expected to mimic those for human PAD; (ii) the model would have relevant human risk factors included, such as older age; (iii) the cost of the model would be low; (iv) the model would be easy to establish and maintain; (v) it would be straightforward to study medical, surgical and endovascular interventions within the model; and (vi) the model would offer sufficient material for the study of downstream cellular and molecular mechanisms.

Most currently employed PAD animal models focus on inducing HLI by surgical methods (Table 3). There are huge variations in the methods employed to induce HLI. There are inconsistencies in the surgical approach used (such as the location of the occlusion), the outcome measurements employed, the choice of animal species and representation of risk factors relevant to the PAD progression in humans (Table 3). In the remainder of this review, the different types of methods employed are discussed and the potential for more appropriate pre-clinical methods.

Table 3
Examples of commonly used pre-clinical animal models involving manipulation of the FA and iliac artery to study lower limb ischemia

Abbreviations: CD, cluster of differentiation; CIA, common iliac artery; EIA, external iliac artery; IHC, immunohistochemistry; IL, interleukin; KK/Ay, KAsukabe spontaneous diabetic agouti mouse; NZW, New Zealand White rabbits; NOD–SCID–IL2R, non-obese diabetic-severe combined immunodeficiency interleukin receptor γ null; Sv, Sendaivirus.

Surgical method used to develop HLI Examples of animal species used Examples of end-point assessment methods used 
Ligation of FA Sv-129/C57BL/6 mice LDPI, diffuse optical spectroscopy, diffuse correlation spectroscopy, visible light oximetry, TOF–MRI, angiography, treadmill testing and IHC [38,55,98,99
 C57BL/6 and BALB/c mice  
 Sv129 mice  
 NZW rabbits  
Excision of FA NZW rabbits LDPI; angiography, histology and IHC [41,100
Ligation and excision of FA C57BL/6 mice LDPI, micro-computed tomography analysis angiography, miRNA expression, protein assays, biochemical analysis, histology and IHC [47,23,57,31,32,33,101108
 Myoglobin-transgenic mice  
 Type 2 diabetes-induced C57BL/6 mice  
 Type 1 diabetes induced CD1 mice  
 KK/Ay diabetic mice  
 Immunodeficient nude C57BL/6 and NOD–SCID–IL2R mice  
 Athymic immunocompromised nude rats  
 Sprague–Dawley rats  
 NZW rabbits  
Occlusion of FA C57BL/6 LDPI, blood perfusion measurement by radiolabelled microspheres, gene expression, biochemical analysis muscle contractile properties, histology and IHC [109112
 Sprague–Dawley rats  
 Wistar rats  
 NZW rabbits  
Ligation of CIA Sprague–Dawley rats LDPI; angiography; muscle oxygen tension, muscle oxygen using a microcatheter oxygen probe and histology [113,114
Ligation of EIA Sprague–Dawley rats LDPI; angiography; biochemical analysis, histology and IHC [115,116
Sodium ricinoleate application on FA NZW rabbits Serial Doppler ultrasound [96
FeCl2 application on the FA Sprague–Dawley rats Histological analysis, pain and thermal threshold [97
FeCl3 application on the FA Yorkshire pigs Near-IR fluorescence microscopy [117
Fibrin-laden thrombus injected into FA Yorkshire pigs Histopathological analysis and angiogram [95
Perivascular silastic collars placement around FA High-cholesterol-fed rabbits Histopathological analysis and imaging [118
Surgical method used to develop HLI Examples of animal species used Examples of end-point assessment methods used 
Ligation of FA Sv-129/C57BL/6 mice LDPI, diffuse optical spectroscopy, diffuse correlation spectroscopy, visible light oximetry, TOF–MRI, angiography, treadmill testing and IHC [38,55,98,99
 C57BL/6 and BALB/c mice  
 Sv129 mice  
 NZW rabbits  
Excision of FA NZW rabbits LDPI; angiography, histology and IHC [41,100
Ligation and excision of FA C57BL/6 mice LDPI, micro-computed tomography analysis angiography, miRNA expression, protein assays, biochemical analysis, histology and IHC [47,23,57,31,32,33,101108
 Myoglobin-transgenic mice  
 Type 2 diabetes-induced C57BL/6 mice  
 Type 1 diabetes induced CD1 mice  
 KK/Ay diabetic mice  
 Immunodeficient nude C57BL/6 and NOD–SCID–IL2R mice  
 Athymic immunocompromised nude rats  
 Sprague–Dawley rats  
 NZW rabbits  
Occlusion of FA C57BL/6 LDPI, blood perfusion measurement by radiolabelled microspheres, gene expression, biochemical analysis muscle contractile properties, histology and IHC [109112
 Sprague–Dawley rats  
 Wistar rats  
 NZW rabbits  
Ligation of CIA Sprague–Dawley rats LDPI; angiography; muscle oxygen tension, muscle oxygen using a microcatheter oxygen probe and histology [113,114
Ligation of EIA Sprague–Dawley rats LDPI; angiography; biochemical analysis, histology and IHC [115,116
Sodium ricinoleate application on FA NZW rabbits Serial Doppler ultrasound [96
FeCl2 application on the FA Sprague–Dawley rats Histological analysis, pain and thermal threshold [97
FeCl3 application on the FA Yorkshire pigs Near-IR fluorescence microscopy [117
Fibrin-laden thrombus injected into FA Yorkshire pigs Histopathological analysis and angiogram [95
Perivascular silastic collars placement around FA High-cholesterol-fed rabbits Histopathological analysis and imaging [118

Methods of HLI induction

A literature search revealed that a number of in vivo models have been used to study PAD. The reported HLI models involve different surgical procedures to induce unilateral ischaemia. The degree of ischaemia obtained using the various surgical procedures depends on the type of surgery, occlusion site and the extent of lower limb artery involved. A brief description of the currently used models of HLI is given below:

Acute ligation and excision model

Currently, the most commonly employed HLI model involves ligation and/or excision of the FA and we will restrict this section to the approaches commonly used in rodents and rabbits (Table 3). This approach is relatively simple to perform, cheap and has well-developed outcome measures. The surgical procedure is performed under continuous infusion of anaesthetics such as isoflurane and the animals generally recover within minutes. HLI has been employed in gaining insights into the efficacy behind a number of novel therapeutic strategies, including cytokines [56], genes [52,53], stem cells [88,89] and nanoparticle drug delivery [90] (Tables 1 and 2). There is large variation in the methods of HLI induction employed in different laboratories which might be one of the reasons behind the variability in the outcomes of the therapies tested.

The severity of experimental ischaemia induced depends on the location and length of artery occlusion and the capacity for arteriogenesis and angiogenesis in the species used as illustrated in Figure 2 [91]. For example, a study assessing six different surgical approaches of HLI induction in mice reported marked differences in the degree of ischaemia induced by each approach [92,93]. The ligation of the FA alone results in milder ischaemia and it was seen that collateral flow continued through the deep FA evidenced by very limited toe discolouration or necrosis [54,92,94]. Since in this technique the collateral circulation remains intact, blood flow to the limb is fully restored and there is minimal functional effect. Ligation and excision of the complete FA and its side branches resulted in distal limb ischaemia with toe necrosis [92]. Severe necrosis of the foot and/or limb paralysis is reported after ligation of the proximal FA and the distal saphenous artery in rodents [91]. The transection of the proximal FA at its bifurcation into the deep FA has been reported to lead to total auto-amputation of the leg [92]. The excision of both the FA and femoral vein is reported to result in sudden effects such as severe ischaemia of the toes, sudden foot and knee necrosis and auto amputation. This approach does not reflect the pathophysiology and clinical presentation of CLI, since in patients, the symptoms are due to arterial obstruction and related complications rather the venous involvement [53,92]. The most commonly reported method of ischaemic induction is the ligation of the FA at two points proximally and distally followed by excision of the intervening segment (Figure 2). Excision of the FA 1 cm below the peritoneal reflection results in a moderate degree of limb ischaemia with no muscle or toe necrosis. Excision of the FA 1 cm above the peritoneal reflection results in severe limb ischaemia with severe muscle necrosis. This could be due to the rapid restoration of blood flow through collaterals when ligation is infrainguinal. The difference in excision points produced marked differences in ischaemic symptoms [77,91]. The choice of the surgical approach is crucial in determining the severity of ischaemia and necrosis [93]. The use of a uniform HLI model would make it easy to assess the repeatability of findings and potentially provide a better means to decide which interventions warrant testing in patients. Additionally, ligation and excision results in sudden and acute changes in the lower limb and does not reflect the pathophysiology of chronic limb ischaemia which is the most common presentation in patients.

Schematic diagram of various techniques employed to induce HLI in animal models
Figure 2
Schematic diagram of various techniques employed to induce HLI in animal models

(A) Detailed schematic diagram of the anatomy of major lower limb arteries of mice. The labelling of the major branches are shown as follows; (1) aorta, (2) common iliac artery, (3) internal iliac artery, (4) external iliac artery, (5) epigastric artery, (6) inguinal ligament, (7) FA, (8) lateral circumflex FA, femoral artery (9) proximal caudal FA, (10) superficial caudal epigastric artery, (11) medial proximal genicular artery, (12) popliteal artery and (13) saphenous artery. Abbreviations: AD, adductor femoris muscle; MH, medial hamstring muscle; QF, quadriceps. (B) A simplified diagram showing various surgical techniques employed to induce unilateral HLI, the consequence and their relation to the presentation of lower extremity ischaemia. (i) Ligation of the FA proximally and distally using silk sutures, (ii) ligation of the FA proximally and distally plus excision of the intervening segment, (iii) ligation and excision of complete FA and its side branches, (iv) ligation of proximal FA and the distal saphenous artery and excision of the intervening segment, (v) ligation and excision of both the FA and the femoral vein.

Figure 2
Schematic diagram of various techniques employed to induce HLI in animal models

(A) Detailed schematic diagram of the anatomy of major lower limb arteries of mice. The labelling of the major branches are shown as follows; (1) aorta, (2) common iliac artery, (3) internal iliac artery, (4) external iliac artery, (5) epigastric artery, (6) inguinal ligament, (7) FA, (8) lateral circumflex FA, femoral artery (9) proximal caudal FA, (10) superficial caudal epigastric artery, (11) medial proximal genicular artery, (12) popliteal artery and (13) saphenous artery. Abbreviations: AD, adductor femoris muscle; MH, medial hamstring muscle; QF, quadriceps. (B) A simplified diagram showing various surgical techniques employed to induce unilateral HLI, the consequence and their relation to the presentation of lower extremity ischaemia. (i) Ligation of the FA proximally and distally using silk sutures, (ii) ligation of the FA proximally and distally plus excision of the intervening segment, (iii) ligation and excision of complete FA and its side branches, (iv) ligation of proximal FA and the distal saphenous artery and excision of the intervening segment, (v) ligation and excision of both the FA and the femoral vein.

The severity of tissue necrosis resulting from HLI is reported to vary among mouse strains due to genetic variability. For example, DBA/1J mice have a higher incidence (50%) of necrosis following HLI than C57Bl/6J (20%) or BALB/c (17%) mice [92]. The degree of ischaemic damage resulting from use of one approach in different mice was variable, ranging from necrosis of toes to total hind limb loss following excision of the FA.

Thrombo-embolic models

The main other type of pre-clinical model used for lower limb ischaemia studies is the thrombo-embolic model (TEM). This model was originally produced by intravascular introduction of fibrin clot from whole pig blood into the lower limb arteries of pigs. The model was used to test the efficacy of mechanical thrombectomy devices [95] (Table 3). The TEM is relatively technically easy to create due to the larger size of pig arteries in comparison with rodents and also due to ease of quantification of intravascular thrombosis and angiographic visualization. A similar in situ aortic thrombosis model employed in rabbits was used to assess the effects of thrombolytic drugs [96]. In this model, the FA of rabbits underwent cannulation and the abdominal aortas were denuded and thrombosis was created in situ using a solution of sodium ricinoleate and thrombin. The advantage of this model is the ease of induction and the ability to study the kinetics of thrombus formation. Painting ferrous chloride (FeCl3) on the FA of rats resulted in a novel thrombus-induced ischaemic pain model reported to mimic human peripheral ischaemic pain [97]. FeCl3 application resulted in intravascular thrombosis, ischaemia and mechanical allodynia in the hind paw along with up-regulation of ischaemia-sensitive markers, such as HIF-1α and VEGF. This animal model of thrombus-induced ischaemic pain may be useful in investigating the pathophysiological mechanisms that underlie ischaemia-associated pain and functional alteration [97]. However, since the thrombosis generated in this model lacks concurrent atherosclerosis, it is more representative of embolization than the presentation of most patients with PAD. A number of groups have reported approaches for induction of limb ischaemia by catheter directed embolization to induce FA occlusion, for example within Lewis rats [119]. This approach preserves tissue integrity and reduces inflammation compared with open surgery and is suggested to be useful in studying the early stages of angiogenesis and arteriogenesis [119]. In order to perform this model advanced skills are required in endovascular techniques and angiography.

Animal species used in HLI models and their relevance to humans

Large and small animals have been used in pre-clinical models of PAD [120]. Usually small animal models, mainly genetically modified mice strains, are preferred due to the availability of transgenic strains enabling assessment of the effects of genetic deficiency or overexpression [121]. The availability of large numbers of knockout and transgenic strains, a fully characterized genome, low maintenance and easy handling and the ability to multiply the breeding stock in limited time, makes it easy to plan experiments in these animals [122] (Table 1).

It has been suggested that the limited baseline collateral network in the mouse, rabbit and pig grossly resembles human vascular anatomy [120]; however, collateral density varies among humans [123] and animal species [124]. Previous observations in various strains of mice highlights the role of genetic variation in controlling the collateral number and capacity for remodelling suggesting employing caution when selecting genetically-modified animals [125,126]. For example, BALB/c mice showed significantly reduced recovery of blood flow after FA ligation compared with C57BL/6 [125,126]. The arterial tree structure also differs between the two species, with BALB/c having fewer pre-existing collaterals [99,127]. Dokun et al. [128] identified that the quantitative trait loci LSq-1 (loss of tissue after ischaemia) on chromosome 7 of the C57BL/6 mouse strain contributes a protective allele to prevent limb necrosis after induction of HLI [128]. Recently, Sealock et al. [129] carried out congenic mapping to refine the loci on chromosome 7 (Candq1) and its candidate genes to create an isogenic strain set with extreme difference in collateral extent to assess their role in ischaemic injury [129]. The candidate gene identified in the refined loci is now designated as determinant of collateral extent-1 (Dce-1). The present study further demonstrated that genetic background-dependent variation in collaterals variation is a major factor underlying differences in ischaemic tissue injury [129]. In short, among the various strains of common laboratory mice, BALB/c is most susceptible to the development of ischaemia as it is reported to have slower recovery after induction of HLI and is more susceptible to tissue necrosis and limb loss after FA ligation [99]. This strain may therefore be most appropriate to use in studies of CLI. On the other hand, C57BL/6 mice might be more appropriate as a model of IC [60] due to the presence of extensive pre-existing collateral circulation [130,131].

Patients with PAD usually have a number of risk factors including smoking, diabetes, hypertension, older age and dyslipidaemia. Few pre-clinical studies have incorporated such risk factors within the animal model employed. Most models used have limited or no atherosclerosis either, since species such as rats and dogs are naturally resistant to atherosclerosis development. Another concern is that the lipoprotein patterns and metabolism in some animal models are different from human, which raises concerns about the relevance of findings within these animal models. Use of mice allows relatively straightforward genetic modification and dietary manipulation, for example to create hyper-cholesterolaemia or hyper-homocystenaemia. Mice can also be exposed to cigarette smoke and physical activity can be limited. In many of the previous pre-clinical studies, the rodents used are often relatively young, whereas in humans, the disease usually manifests at advanced age (Table 3). Another drawback of using healthy young animals for FA ligation models is that collateral formation occurs rapidly in younger animals in contrast with PAD patients [91]. These limitations can potentially be overcome by performing studies within pre-clinical models that incorporate some or all of the risk factors common in patients.

Common outcome assessments in PAD models and the relevance to outcomes in patients

Patients with PAD usually seek medical attention in order to improve their ability to mobilize, limit limb pain and because of concerns regarding subsequent amputation. Patients are assessed by a combination of investigations including haemodynamic examinations, such as ABI (ankle brachial index), and anatomical imaging such as computed tomography or conventional angiography. Patients involved in clinical trials may also undergo a number of functional assessments including treadmill walking tests and corridor walking investigations, such as the 6-min walk test, when they present with IC. In patients with CLI, usually the focus is on limb salvage and pain reduction [132,133]. Ideally outcome measures in pre-clinical models would be comparable to those applied in patients where feasible.

However, in most pre-clinical models of PAD, outcomes are assessed by examining limb perfusion and using imaging based or histological assessments of angiogenesis (Table 4). Some of the advantages and disadvantages of the most commonly used outcome assessments employed in animal models of PAD are listed below with emphasis on how the outcome is assessed and graded, how reproducible and feasible the assessment is and how relevant the endpoints are to those used in patients.

Table 4
Commonly used outcome measures in pre-clinical models of PAD

Abbreviations: CT, cell therapy; GT, gene therapy; NOD SCID, non-obese diabetic severe combined immune deficient; NZW, New Zealand White rabbits; RT, recombinant protein therapy; Sv, Sendaivirus.

Methods of outcome assessment End-point Therapy tested Animal used for HLI induction 
LDPI Improved blood supply GT, RT C57BL/6 mice [53,23
  CT BALB/c-nu/nu mice [53,37
  RT Sv129 mice [55
  CT NOD SCID mice [32,58
  GT, RT NZW rabbits [47,56,57
Angiogram Improved blood supply; neoangiogenesis; arteriogenesis GT, RT, CT NZW rabbits [4143,45,4749,56,57,35
Contrast-enhanced ultrasound Improved blood supply; aerobic capacity; exercise tolerance; improved limb function GT NZW rabbits [38,39,43,46
Contrast-enhanced MRI Improved blood supply; therapeutic angiogenesis GT NZW rabbits [38,40,42
Intra-arterial Doppler guidewire Improved blood supply; increased blood pressure in ischaemic limb GT NZW rabbits [45,47
Fluorescent microspores Improved blood supply; EPC recruitment GT NZW rabbits [42
  RT C57BL/6 mice [23
  CT NOD SCID mice [58
Frame count cine densitometry Improved blood supply GT NZW rabbits [41
31P-magnetic resonance spectroscopy Improved blood supply; aerobic capacity; exercise tolerance; improved limb function GT NZW rabbit [46
Limb muscle gene expression Increased angiogenic growth factor gene expression leading to increased HIF-1α and VEGF protein; therapeutic angiogenesis GT, CT NZW rabbits [44,48,52,54,30,37
  GT, CT BALB/c mice [52,37
  GT Streptozotocin-induced type-1 diabetic mice [54
  CT Dogs [30
Limb muscle immunohistochemistry Increased collateral size; increased collateral and capillary densities, increased expression of eNOS and Ki67 proliferative markers; decreased number of necrotic cells and interstitial fibrosis GT C57BL/6 mice and of BALB/c nu/nu mice [42,53
  GT, CT Diabetic mice [54,31
  CT NOD SCID mice [32,58
  GT, RT, CT NZW rabbits [39,43,44,47,34,57,35
  GT Sprague–Dawley rats [50
  CT Dogs [30
Modified miles assay Improved vascular permeability GT NZW rabbits [39,40,42,46
Calf blood pressure ratio Improved blood supply; arterial remodelling; neoangiogenesis GT NZW rabbits [44,47,49
Isometric gastrocnemius muscle contraction Altered muscle functioning by assessing muscle force and muscle mass GT BALB/c mice [52
Visual scoring of limb Improved blood supply; visual improvement; limb salvage RT Sv129 mice [55
  GT BALB/c mice [52
Methods of outcome assessment End-point Therapy tested Animal used for HLI induction 
LDPI Improved blood supply GT, RT C57BL/6 mice [53,23
  CT BALB/c-nu/nu mice [53,37
  RT Sv129 mice [55
  CT NOD SCID mice [32,58
  GT, RT NZW rabbits [47,56,57
Angiogram Improved blood supply; neoangiogenesis; arteriogenesis GT, RT, CT NZW rabbits [4143,45,4749,56,57,35
Contrast-enhanced ultrasound Improved blood supply; aerobic capacity; exercise tolerance; improved limb function GT NZW rabbits [38,39,43,46
Contrast-enhanced MRI Improved blood supply; therapeutic angiogenesis GT NZW rabbits [38,40,42
Intra-arterial Doppler guidewire Improved blood supply; increased blood pressure in ischaemic limb GT NZW rabbits [45,47
Fluorescent microspores Improved blood supply; EPC recruitment GT NZW rabbits [42
  RT C57BL/6 mice [23
  CT NOD SCID mice [58
Frame count cine densitometry Improved blood supply GT NZW rabbits [41
31P-magnetic resonance spectroscopy Improved blood supply; aerobic capacity; exercise tolerance; improved limb function GT NZW rabbit [46
Limb muscle gene expression Increased angiogenic growth factor gene expression leading to increased HIF-1α and VEGF protein; therapeutic angiogenesis GT, CT NZW rabbits [44,48,52,54,30,37
  GT, CT BALB/c mice [52,37
  GT Streptozotocin-induced type-1 diabetic mice [54
  CT Dogs [30
Limb muscle immunohistochemistry Increased collateral size; increased collateral and capillary densities, increased expression of eNOS and Ki67 proliferative markers; decreased number of necrotic cells and interstitial fibrosis GT C57BL/6 mice and of BALB/c nu/nu mice [42,53
  GT, CT Diabetic mice [54,31
  CT NOD SCID mice [32,58
  GT, RT, CT NZW rabbits [39,43,44,47,34,57,35
  GT Sprague–Dawley rats [50
  CT Dogs [30
Modified miles assay Improved vascular permeability GT NZW rabbits [39,40,42,46
Calf blood pressure ratio Improved blood supply; arterial remodelling; neoangiogenesis GT NZW rabbits [44,47,49
Isometric gastrocnemius muscle contraction Altered muscle functioning by assessing muscle force and muscle mass GT BALB/c mice [52
Visual scoring of limb Improved blood supply; visual improvement; limb salvage RT Sv129 mice [55
  GT BALB/c mice [52

Assessment of ischaemia and limb function

Many studies using the unilateral HLI model have reported outcomes based on scoring systems. A ‘clinical score’ calculation based on the number of necrotic toes is suggested to be useful in assessing the recovery from ischaemia achieved by different therapies and is reported to have low inter-observer variability [91]. The severity of tissue necrosis in the ischaemic limb has been previously assessed using the following scale: 0=no necrosis, 1=one toe, 2=2 or more toes, 3=foot necrosis, 4=leg necrosis and 5=auto amputation of the entire leg [93]. A more elaborate scoring system developed by Chalothorn et al. [127] called ‘index of ischaemia’ or ‘foot appearance’ ranged from 0 to 11 [0=normal, 1–5=cyanosis or loss of the nail(s), the score representing the number of affected nails; 6–10=partial or complete atrophy of the digit(s), the score reflecting the number of affected digits; 11=partial atrophy of the fore foot]. In a subsequent study, the same authors presented a more simplified ‘index of ischaemia’ as follows: 0=normal, 1=cyanosis or loss of the nail(s), 2=partial or complete atrophy of the digit(s) and 3=dry necrosis beyond the digits into the front part of foot [134].

A commonly used functional scoring system for the usage of the hind limb represents an index of muscle function, represented as 0=normal; 1=no toe flexion; 2=no plantar flexion and 3=dragging of the foot [135]. Chalothorn and Faber [134] developed another scoring system for muscle function, named ‘clinical use score’, which grades the ischaemic hind limb usage as follows: 0=normal toe and plantar flexion; 1=no toe but plantar flexion; 2=no toe or plantar flexion and 3=dragging of the foot. The use of these scoring systems varies in different studies and in many cases the inter-observer variation in assessment is not reported. Furthermore, even though the assessment of the gross appearance and the functional defects are considered in these types of scoring systems, it should be noted that the scores are subjective. Ideally they should be reported by someone blinded to any intervention being assessed.

Measurement of perfusion

Most studies of the unilateral HLI model report perfusion within the operated limb in comparison with the un-operated contralateral limb. The most common technique for assessing blood flow is laser Doppler perfusion imaging (LDPI; Table 4). This non-invasive technique is used widely to monitor the effect of surgical interventions, drugs or cell therapies on tissue perfusion. LDPI has been successfully used to monitor angiogenesis responses after therapeutic interventions within HLI models in rodents [47,101,110,136]. LDPI enables real-time assessment of microcirculation throughout the time frame imaged.

Muscle oxygenation can be measured by transcutaneous oxygen pressure (TcPo2) using a modified Clark electrode [137]. In pre-clinical models, an incision is made to insert the electrode into the lower limb muscle to measure the partial pressure of oxygen [138]. This method allows for reproducible measurement, avoids cutaneous blood flow and it is not significantly temperature-dependent.

Laser speckle flowmetry (LSF) is a recently adapted quantitative method for measuring relative blood velocity as well as shear stress in large microvascular networks. The advantage of LSF over the LDPI is its better resolution and the ease of processing [129]. The combination of LSF and intravital microscopy preparations of the microvasculature aids in determining the relative and absolute haemodynamics or blood flow within a wider region in the limb, while maintaining the resolution of the individual microvessel level [129]. A recent modification of the LSF, the transillumination-LSF, was used to report the first direct measurements of regional shear stress changes and direction in collateral arteries of mice [139]. One major disadvantage of the LSF in microvascular research is that it is considered to be blind to native collaterals [140]. At resting state, a healthy tissue is considered to have limited or no pressure drop and collateral flow [141], thus since LSF cannot assess the collateral flow until a severe obstruction is induced, its use in understanding the microvascular haemodynamics in models without induction of shear stress changes is questionable.

The standardization of anaesthesia dose, maintenance of a constant body temperature and blood pressure combined with currently available high-resolution LDPI provides safe, accurate and reproducible perfusion assessment of the entire limb [120,142]. However, blood flow assessed in this way is not a parameter usually tested in the clinical setting where ankle brachial pressure index (ABPI) is more commonly employed. Even though the effect of improvement in blood flow following a therapeutic intervention provides value in assessing the efficacy of an intervention, additional clinically relevant parameters should ideally be assessed.

Angiography

Visualization of collateral vessels using angiography by radiographic equipment employing contrast agents is a standard imaging technique used in HLI models [143]. The majority of the investigators have used angiography for the qualitative assessment of vessel growth within HLI models [144,145] (Table 4). A few studies have attempted to report the blood vessel density as the percentage of pixels per digitized image within rodents [143,146] or have quantified neovascular responses by counting the crossing points of arterial branches at perpendicular lines [147]. Some studies have attempted to develop an angiographic score using a composite of 2 mm2 grids printed on a transparent sheet placed above the medial thigh area on each film [49,148,149]. The angiographic score is then calculated for each angiogram as the ratio of grid intersections crossed by the contrast material: opacified arteries divided by the total grid intersections within the medial thigh area [149].

The main advantage of angiography is the better resolution achieved compared with LDPI. It is also fast and is practical for experiments involving larger sample numbers. In spite of these advantages, angiography is less popular for a number of reasons, including the reduced accuracy of collateral blood flow measurement and the lack of radiographic spatial resolution [91]. A major drawback of angiography is its invasive nature, hence typically post-mortem angiography and grading of collateral filling is performed. Another major drawback of angiography is that the viewing angles are pre-determined and limited in number. Additionally, the superimposition of blood vessel makes it impossible to distinguish an overlapping vessel from a true side branch. Thus, it is often impossible to distinguish between the microvessels undergoing angiogenesis from arteriogenesis. Furthermore, conventional projection angiography cannot generate 3D data and hence it cannot determine the spatial relationship between the vasculature and the surrounding tissues. Also, angiography requires X-ray exposure which requires special equipment, licensing and appropriate radiation protection methods. Moreover, the technique employs sophisticated instruments which are technically demanding and labour intensive. Angiograms are used in the assessment of patients although this is primarily to assess the feasibility of interventions not to assess collateral presence.

Computer tomography

2D multi-detector row computer tomography (CT) angiography and 3D volume rendering for depiction of patterns of arterial growth, quantification of blood vessel density and volume, have previously been successfully employed for the visualization of arterial collateral networks in rodents [150]. CT angiography has been performed with the injection of barium sulfate suspension and laboratory-grade latex as vascular contrast agents. Latex is injected into the hind limbs through a catheter inserted proximal to the aortic bifurcation and it stabilizes the barium sulfate suspension and allows subsequent dissection of the specimen [150]. Subsequent to the angiography, silver staining was performed which confirmed that the contrast agent filled both small arteries (>188 μm) and surrounding arterioles (10–40 μm). The digitally-subtracted 2D CT angiographic sections are then quantified and subsequently 3D images are reconstructed.

Another novel method of collateral formation assessments is microCT (μCT) for the 3D anatomic study of post-ischaemia neo-vascularization [151]. μCT reconstruction and quantification of arteriolar number as low as 16 μm resolution has recently been performed within the thigh and calf of mice subjected to FA occlusion [152]. In this approach, the abdominal aorta is cannulated and perfused with papaverin hydrochloride to obtain maximal vasodilation, followed by perfusion with contrast agents. The morphology of the hind limb vascular network is imaged and analysed with a high resolution μCT imaging system. The quantitative analysis of μCT images can provide the total number of vascular structures in consecutive z-axis slices. Subsequent histological analysis has been reported to show comparable arteriolar densities to the assessment made by μCT imaging [152]. Thus, 2D and 3D multi-detector row μCT angiography has emerged as a potential tool in the PAD pre-clinical experimental settings.

The major advantage of this technique is that the 3D multi-detector row CT angiograms can be rotated for the exclusion of blood vessel foreshortening and also for discriminating the pre-existing collateral vessels and the newly formed vessels [150]. The 3D anatomical information can be supplemented with the limb perfusion measurements from LDPI [150] or contrast-enhanced MRI to obtain regional and muscle-specific perfusion measures [153]. The major limitation which prevents the wide use of these techniques is the requirement for expensive instruments and necessity of dedicated rooms and trained personnel. Another possible limitation of these techniques is the possibility of development of allergic reactions to the contrast agents [91].

Histopathology

Histopathological assessment is the most common technique used to understand pathological changes within HLI models. Many studies suggest the correlation between histological assessment and altered perfusion rates assessed by LDP1 [101,143]. Immunohistochemical methods are used to quantify the ECs as an index of capillary density within ischaemic limbs. The contra-lateral limb is used for normalization. A monoclonal antibody against von Willebrand factor (vWF) is generally used as a powerful EC marker because this molecule is constitutively expressed and its expression does not depend on phenotypic changes [149].

The advantages of histopathology are the ability to be combined with immunohistochemistry or in situ hybridization to localize proteins at the cellular level. The most-commonly assessed histological measurement of perfusion within the HLI models is capillary density measurement. Angiogenesis is often assessed as the capillary to fibre ratio within the gastrocnemius or gracialis muscles [126,154,155]. One issue with using the contralateral limb as a control is that assessments within this limb may be influenced by angiogenic factors released into the systemic circulation from the ischaemic limb (remote pre-conditioning) [91]. However, researchers have tried to overcome this shortcoming by normalizing the findings in the ischaemic limb to the contralateral control limb examined simultaneously.

FUTURE: DEVELOPMENT OF AN IDEAL PAD MODEL

Development of better animal models is potentially important to improve translation of pre-clinical findings. A number of potential improvements could be made including:

  • 1) Better simulating the pathophysiology of the most common presentations of PAD such as IC and CLI;

  • 2) Incorporation of more representative pathology, such as concurrent atherosclerosis;

  • 3) Incorporation of common risk factors present in patients, such as older age, diabetes and smoking;

  • 4) Use of outcome assessments common in human trials which include functional assessments.

Below some of the problems with current animal models of PAD are further discussed and solutions suggested.

Use of methods to induce more gradual onset of ischaemia

The commonly used HLI models employ acute arterial occlusion which does not replicate accurately the chronic and established ischaemia which is present in most patients presenting with PAD. Ligation of the FA in the absence of pre-existing lower limb arterial stenoses leads to sudden changes in haemodynamic and pressure gradients within collateral arteries. These sudden changes lead to remodelling and growth of conductance vessels promoting the formation of a collateral circuit [156]. It has been shown previously that the sudden changes in fluid shear stress induce changes in the gene expression through shear response elements promoting collateral artery enlargement and arteriogenesis [157,158]. It has also been shown that shear stress and inflammatory genes play critical roles in collateral artery enlargement [159161]. The ECs are profoundly influenced by shear stress which induces post-translational modifications of proteins and gene expression [162]. The HLI models appear to simulate the pathophysiology of acute ischaemia best. Models of more gradual and progressive onset of ischaemia are needed to simulate the more common presentation of IC and CLI.

Previous reports suggest that ameroid constrictors can be used to induce a gradual progressive stenosis [163,164]. This approach has been used to generate gradual onset of ischaemia in rabbits, rats and mice [109,114,165]. When implanted in the body, the inner diameter of the ameroid constrictor narrows gradually because of the hygroscopic property of the casein plastic material used to coat the inner surface. In a study monitoring gradual arterial occlusion induced by an ameroid constrictor within C57BL/6 mice [109] using LDPI, the blood flow reached the lowest level on post-operative day 14. By post-operative day 35, the blood flow recovery level was lower than that commonly measured within acute HLI models [109]. The expression levels of several genes crucial for the response to ischaemia within the gastrocnemius muscle was different in this model than seen within acute HLI models. The extensive muscle necrosis seen within acute models was not reported. The pressure gradients across the collateral arterial beds were low and endothelial nitric oxide synthase (eNOS) and early growth response (EGR)-1 genes were not activated to induce collateral artery enlargement. These findings suggest that this approach may be more representative of the most common presentations of patients with PAD [109].

Location of arterial stenosis or occlusion

The surgical method and the location of occlusion or stenosis determine the severity as well as the region undergoing ischaemic changes in the limb. Hence, it is crucial that the surgical method as well as location to induce HLI is standardized (Figure 2). The arterial segment selected to induce occlusion varies in different studies and includes the iliac, deep femoral, superficial femoral, saphenous and popliteal arteries. In some cases, both the FA and the vein are occluded. The distal ligation of the FA alone results in mild ischaemia [92]. Proximal ligation of the FA leads to more severe ischaemia, since the blood flow in the deep FA is also interrupted [92]. Goto et al. [92], who assessed six different methods of ischaemia induction involving the FA, reported that milder ischaemia can be created by excising the FA just below the deep FA bifurcation. A more severe and stable ischaemia can be induced by excising the FA from the distal site of the bifurcation of the deep FA into the saphenous artery [92] (Figure 2Biv). A severe ischaemia model was developed in rabbits and mice with ligation and excision of the external iliac artery and excision of the entire FA [101,166,167]. Thus, it is evident that the severity of ischaemia induced by these various methods is not uniform, hence the comparison of therapeutic results obtained by using these models is not straightforward. Milder ischaemia models would seem most appropriate to study interventions for IC. More severe ischaemia models would seem most suited to studies on CLI.

Clinically-relevant functional assessment and timing of assessments

Most studies reporting the effect of interventions in pre-clinical models of PAD examine their effect in terms of LDPI. Functional assessments used in PAD patients include walking times on treadmills, the 6-min walk test, physical performance assessments and monitoring of physical activity for extended periods, for example using pedometers or accelerometers [168,169]. In clinical trials, patients with IC are primarily assessed for improvement in pain-free walking capacity on treadmills or in corridor walking tests [170174], whereas patients with CLI are assessed in terms of limb pain and salvage. Hence, it appears appropriate that PAD pre-clinical animal models incorporate similar outcome measures.

Some drugs which have previously been reported to improve the recovery of perfusion to the limb in HLI models have failed to improve treadmill walking times in clinical trials within IC patients [173,175,176]. A Scottish–Finnish–Swedish PARTNER study, which included 111 CLI patients, could not demonstrate any statistically significant benefit of taprostene (an analogue of prostacyclin which inhibits platelet aggregation) over placebo [177].

Developing methods to assess similar outcomes in pre-clinical PAD models would appear appropriate in order to better identify novel interventions to test in patients. In this regard, a voluntary wheel-running test has been used to determine physical activity in a HLI mice model [178]. Also, treadmill endurance exercise tests have been used in a few pre-clinical studies [178,179]. Some investigators have studied the gait of mice and shown these to be altered in response to limb ischaemia, as has been reported in PAD patients [180184]. Assessing appropriate PAD models using voluntary running wheels, miniature treadmills and computerized gait assessment devices may provide a more effective way of identifying novel therapies for PAD.

CONCLUSION

The use of pre-clinical animal models is potentially indispensable to identify novel therapies for PAD. As described in this review there is however significant scope for more relevant approaches to these studies including the model used, the design of the study and the outcomes assessed. Single investigations alone may also be inadequate to guide decisions about which interventions move forward to studies in PAD patients. Systematic reviews or meta-analysis of animal studies have been proposed as additional means to best select approaches that are likely to have translation potential [185]. Earlier, the concept of ‘co-clinical’ trials have been introduced in the field of cancer, in which the efficacy of a candidate drug is tested in parallel in both human and pre-clinical rodent models, which requires animal models closely mirroring the human counterpart and the two studies are closely aligned [186,187]. It is hoped by applying these types of approaches greater success in identifying new therapies for PAD will be achieved. It is accepted that novel therapies are badly needed to advance the outcome of this patient group.

We apologize to our colleagues for the omission of certain references to many important contributions due to space limitations. The funding bodies played no role in generation of the data presented in this publication.

FUNDING

This work was supported by the National Health and Medical Research Council [grant numbers 1079369, 1079193, 1063476, 1021416, 1000967 and 1019921 (to J.G.)]; the Queensland Government (to J.G.); the Townsville Hospital Private Practice Trust; the Research Infrastructure Block Grant; and the Medicine Incentive Grant, School of Medicine, James Cook University (to S.M.O.); the College of Medicine (to S.M.O.); the Queensland Research Centre for Peripheral Vascular Disease (to S.M.O.); and the Centre of Research Excellence (to S.M.O.).

Abbreviations

     
  • ABI

    ankle brachial index

  •  
  • ADRC

    adipose-derived regenerative cell

  •  
  • CLI

    critical limb ischaemia

  •  
  • CT

    computer tomography

  •  
  • Del-1

    development-regulated endothelial locus-1

  •  
  • EC

    endothelial cell

  •  
  • eNOS

    endothelial nitric oxide synthase

  •  
  • EPC

    endothelial progenitor cell

  •  
  • FA

    femoral artery

  •  
  • FGF

    fibroblast growth factor

  •  
  • HIF

    hypoxia-inducible growth factor

  •  
  • hiPSC

    human-induced pluripotent stem cell

  •  
  • HLI

    hind limb ischaemia

  •  
  • IC

    intermittent claudication

  •  
  • LDPI

    laser Doppler perfusion imaging

  •  
  • LSF

    laser speckle flowmetry

  •  
  • MCP

    monocyte chemoattractant protein

  •  
  • MSCs

    mesenchymal stem cells

  •  
  • PAD

    peripheral arterial disease

  •  
  • PDGF

    platelet-derived growth factor

  •  
  • RAVE

    Regional Angiogenesis with VEGF in PAD

  •  
  • TEM

    thrombo-embolic model

  •  
  • VEGF

    vascular endothelial growth factor

  •  
  • vWF

    von Willebrand factor

  •  
  • μCT

    microCT

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