Recent developments in targets for ischemic foot disease

Diabetes is a key risk factor for ischaemic foot disease, which causes pain, tissue loss, hospital admission, and major amputation. Currently, treatment focuses on revascularisation, but many patients are unsuitable for surgery and revascularisation is frequently unsuccessful. The authors describe recent research in animal models and clinical trials investigating novel medical targets for ischaemia, including theories about impaired wound healing, animal models for limb ischaemia and recent randomised controlled trials testing novel medical therapies. Novel targets identified in animal models included stimulating mobilisation of CD34+ progenitor cells through upregulating oncostatin M or microRNA‐181, downregulating tumour necrosis factor superfamily member 14, or activating the Wingless pathway. Within the ischaemic limb vasculature, upregulation of apolipoprotein L domain containing 1, microRNA‐130b or long noncoding RNA that enhances endothelial nitric oxide synthase expression promoted limb blood supply recovery, angiogenesis, and arteriogenesis. Similarly, administration of soluble guanylate cyclase stimulators riociguat or praliciguat or 3‐ketoacyl‐CoA thiolase inhibitor trimetazidine promoted blood flow recovery. Translating pre‐clinical findings to patients has been challenging, mainly due to limitations in clinically translatable animal models of human disease. Promising results have been reported for administering plasmids encoding hepatocyte growth factor or intra‐arterial injection of bone marrow derived cells in small clinical trials. It remains to be seen whether these high resource therapies can be developed to be widely applicable. In conclusion, an ever‐expanding list of potential targets for medical revascularisation is being identified. It is hoped that through ongoing research and further larger clinical trials, these will translate into new broadly applicable therapies to improve outcomes.

Ischaemia is the key risk factor for the failure of diabetes-related foot ulceration healing and increases the risk of hospital admission and major amputation. 1 Together with ischaemic gangrene and ischaemic rest pain, ischaemic ulceration constitutes one of the presentations of chronic limb threatening ischaemia (CLTI).The treatment of CLTI focuses on revascularisation by open or endovascular surgery along with treatment of associated infection and wound debridement as recommended by global guidelines. 1,2Revascularisation is the main treatment of CLTI but is not always possible or successful with minor and major amputation still being a common outcome, thus highlighting the need for identifying additional treatments. 1,3This narrative review discusses the pathological factors driving impaired wound healing and the findings from recent animal model studies and clinical trials to develop new therapies for CLTI and ischaemic wounds.

| ANIMAL MODELS OF FOOT ISCHEMIA
Animal studies of peripheral artery disease (PAD) employ surgically induced hind limb ischaemia (HLI) (Supplement Figure 1). 4,5Most studies have used surgical ligation or excision. 5,6Less commonly, artery occlusion was achieved by cautery or injecting particles into the artery (Supplement Figure 1). 7,8Photochemical induction of thrombotic artery occlusion has also been used. 9Mice are the most commonly used animal models due to their low cost and the availability of a range of genetic tools to study the effects of individual pathways.Other species used include rats, rabbits and pigs. 5Mouse strains vary in terms of their susceptibility to ischaemia induction, with C57BL/6 being relatively resistant whilst BALB/c being very sensitive. 10In BALB/c mice, proximal as opposed to distal artery occlusion causes more severe ischaemia. 11In C57BL/6 mice, which is a common background for most genetically modified models, excision of the femoral artery causes a rapid reduction in limb blood supply, which recovers to normal within 4 weeks (Supplement Figure 2). 6acement of ameroid constrictors around the femoral artery causes gradual femoral artery narrowing over 3-5 days, and it is believed to cause less rapid increase in shear stress, more limited induction of growth factors and less severe mobilisation of inflammatory cells that limits blood flow recovery. 12This model was recently extended by a second stage procedure performed 2 weeks after ameroid placement when the femoral artery and any collateral arteries were excised (Supplement Figure 2). 6This two-stage procedure leads to prolonged severe ischaemia and impaired ambulation not evident after femoral excision alone, better simulating the human presentation of PAD (Supplement Figure 2). 6It should be noted, that all these animal models have multiple limitations in simulating human PAD.For example, ischaemia induced in these animal models does not result from atherosclerosis, as in humans.Furthermore, there are multiple pathophysiological differences between humans and rodents.As a result, translating findings from animal models to patients remains challenging.

| Rewiring macrophage polarisation
Ischaemia drives an inflammatory response that contributes to tissue perfusion recovery.Macrophages, which produce growth factors for vascular remodelling, are essential for this process.Uncontrolled macrophage activation is implicated in delayed wound healing and thus manipulation of macrophages has been proposed to promote wound healing.Iron overload has been implicated in ischaemia associated with renal failure and induces a pro-inflammatory M1 activation state in macrophages, triggering senescence in resident fibroblasts and impairing wound healing. 13,14A study using a HLI mouse model found that the lipid mediator resolvin D1, acting through its receptor ALX/FPR2 on macrophages, promotes perfusion recovery. 15Tissue macrophages can be instructed to resemble tumour-associated macrophages, thereby helping in suppressing inflammation, stimulating angiogenesis, and activating fibroblasts, ultimately leading to an improvement in diabetic wound healing. 16 a study conducted on 60 individuals without diabetes and 53 patients with type 2 diabetes, we examined monocyte subsets based on CD (cluster of differentiation) 14 and CD16 markers and char- ) monocytes based on in vitro phenotypes. 17tients with diabetes showed an imbalanced M1/M2 ratio, primarily due to a reduction in M2 monocytes.The M1/M2 ratio was correlated with waist circumference and HbA1c levels.Among diabetic patients, the decrease in M2 monocytes and increase in the M1/M2 ratio were specifically associated with microangiopathy.
Additionally, there was a decrease in M2 monocytes in the bone marrow of diabetic patients, with a relative excess of M2 cells in the bloodstream.Stimulation of the bone marrow with granulocytecolony stimulating factor mobilised M2 macrophages in diabetic individuals but not in healthy individuals.Thus, the study demonstrates that type 2 diabetes leads to a significant reduction in antiinflammatory M2 monocytes, primarily due to dysregulation in bone marrow function.This defect may have a negative impact on microangiopathy, suggesting that the imbalance in monocyte polarisation may contribute to the development of complications in distant organs, including the wound sites. 17To understand the implications of these findings in the diabetic ischaemic wound healing process, it should be emphasised that M2 macrophages, which are defective in diabetes, are directly implicated in angiogenesis and responses to ischaemia.M2 macrophages secrete various factors and cytokines that promote angiogenesis.In addition to growth factors, M2 macrophages produce enzymes, such as matrix metalloproteinases that facilitate the remodelling of the extracellular matrix, allowing endothelial cells to migrate and form new vessels.
They also secrete cytokines such as interleukin-8 (IL-8) and tumour necrosis factor-alpha that promote endothelial cell proliferation and vessel sprouting.Furthermore, M2 macrophages can directly interact with endothelial cells through cell-cell contact and the release of adhesive molecules.These interactions help in the stabilisation and maturation of newly formed blood vessels. 18morbidities often observed in diabetes, such as dyslipidemia, can also contribute to an imbalance in monocyte phenotype and can sustain the ischaemic component of delayed wound healing.In individuals with familial and non-familial (NFH) hypercholesterolaemia, compared to those with normocholesterolemia (CTRL), higher levels of circulating pro-inflammatory M1 monocytes were detected, marked by the CD68(þ)CCR2(þ) phenotype.Additionally, NFH individuals showed reduced anti-inflammatory M2 monocytes, characterised by the CX3CR1(þ)CD163(þ)/CD206(þ) phenotype.The M1/M2 ratio was increased in hypercholesterolemic individuals and correlated with pre-treatment low density lipoprotein-cholesterol levels, and was strongly associated with the severity of atherosclerosis.This study suggests that human hypercholesterolaemia is linked to an imbalance of circulating monocytes, favouring a proinflammatory state.This imbalance can potentially contribute to the development of atherosclerosis, thereby sustaining the ischaemic component of delayed wound healing. 19ng and colleagues analysed publicly available transcriptomics data and found downregulation of period circadian regulator 1 gene expression in monocytes from patients with CLTI compared to controls. 20In C57BL/6 mice, an adenovirus delivering mRNA expressing period circadian regulator 1 significantly improved recovery of blood flow following femoral artery excision.This effect was associated with a switch in macrophage phenotype from M1 to M2 and increased capillary density.It should be noted that the role of monocytemacrophage phenotype may be different in men and women.Oestrogen treatment can protect against the inflammatory response induced by M1 stimuli in macrophages, whereas the loss of oestrogen during menopause is associated with impaired M2 activation. 21tably, targeting macrophage polarisation has recently reached clinical application.A novel compound (ON101) that inhibits proinflammatory M1 macrophages and stimulates adipose precursor cells to secrete granulate colony stimulating factor and CXCL3 to increase anti-inflammatory M2 macrophages was tested in a multicentre, phase 3 randomized clinical trial. 22Among 236 patients with diabetes and Wagner grade 1 or 2 foot ulcers, those randomized to ON101 cream had an increased rate of healing (odds ratio 2.84) than those in the comparator group.There is a great expectation that new therapeutics directed to rewiring macrophage polarisation will also aid healing of ischaemic wounds, but to date there is no definite clinical demonstration.

| Disarming neutrophils
Neutrophils can interact with macrophages to impair wound resolution.Neutrophils release web-like structures called neutrophil extracellular traps (NETs) to trap and kill pathogens (NETosis).
Neutrophil extracellular traps act as a physical barrier and contain antimicrobial components that neutralise and immobilise microbes, thereby aiding in host defence against infections.The role of NETs in the healing of DFUs has been examined in a growing number of studies in the last 10 years, making it a current hot topic in this research field. 23A study demonstrated that neutrophils from diabetic individuals and mice are primed to produce NETs. 24Higher levels of NETs and delayed wound healing were observed in diabetic mice, but these effects were mitigated in mice lacking peptidylarginine deiminase 4 (PAD4).Additionally, disrupting NETs with DNase 1 accelerated wound healing in both diabetic and non-diabetic mice.These findings suggest that inhibiting NETosis could be a potential strategy to improve wound healing in diabetes. 24Some of the mechanisms that sustain NET release have been clarified, including oxidative stress, epigenetic changes driven by microRNAs and activation of the inflammasome. 25We found that, in patients with diabetes, neutrophils are prone to release NETs.Specifically, NET components were enriched in samples collected from non-healing DFUs, and high levels of neutrophil elastase in the wounds were associated with infection and worsened ulcer outcomes. 26Furthermore, inhibiting the activity of PAD4, an enzyme involved in NETosis, improved wound healing in diabetic mice. 26The observation that NET components are overrepresented in non-healing diabetic wounds has been confirmed by others, supporting this as a therapeutic target. 26,27cently, blocking NETosis was reported to accelerate wound healing in mice by reducing endothelial-to-mesenchymal transition in the microcirculation and promoting angiogenesis. 28This opens the possibility that NETosis is also involved in delaying wound healing in the presence of ischaemia and that, consequently, targeting NETosis is a strategy to accelerate vascular recovery.This area of research is still preliminary, as the role of NETosis in angiogenesis may vary according to the disease setting.In pulmonary arterial hypertension, NETs appear to be involved in the stimulation of angiogenesis and blocking NET release was proposed as a therapy. 29NETing cells can also inhibit angiogenesis by secreting neutrophil elastase and αdefensins, which generate molecules that inhibit blood vessel formation and inactivate proangiogenic factors.It should be noted that a circulating proangiogenic neutrophil subpopulation has been recently discovered in both mice and humans, which is rapidly recruited to oxygen-deprived tissues by vascular endothelial growth factor A. 30,31 How NETosis interacts with the pro-or anti-angiogenic function of neutrophils remains to be elucidated.

| TARGETS FOR TREATING LIMB ISCHEMIA FROM FINDINGS OF RECENT RODENT STUDIES
Figure 1 and Table 1 summarise recent findings of targets that significantly impacted HLI in mice models.

| Promoting mobilisation, homing and differentiation of bone marrow derived cells
A long-standing concept in recovery from limb ischaemia has been that bone marrow-derived cells are mobilised in response to HLI and GOLLEDGE ET AL.
-3 of 12 recruited to ischaemic tissue in response to local growth factors, where they promote new capillaries formation (angiogenesis) and remodelling and expansion of existing collateral arteries (arteriogenesis) (Figure 1). 3 A range of growth factors such as stromal cellderived growth factor-1α (SDF1α), have been shown in rodent models to promote mobilisation of progenitor cells from the bone marrow and promote increased density of limb capillaries. 32A study in C57BL/6 mice with HLI using single-cell RNA sequencing suggested that CD34þ progenitor cells were recruited to ischaemic muscles where they differentiate into fibroblasts and promote angiogenesis via the oncostatin M-angiopoietin-like protein pathway. 33Oncostatin M blocking antibody or CD34þ cell deficiency significantly reduced blood flow recovery in C57BL/6 mice with HLI. 33The role of this pathway is controversial because other studies demonstrated that blocking oncostatin M reduced the excess inflammatory myelopoiesis seen in diabetes, rescued bone marrow stem cell mobilisation, improved stem-cell transfer to ischaemic sites and facilitated recovery of limb blood supply. 34u and colleagues identified that tumour necrosis factor superfamily 14 (TNFSF14) inhibited endothelial progenitor cell (EPC) mobilisation by downregulating SDF-1ά, significantly worsening limb blood flow in C57BL/6 mice after HLI. 32In vitro, TNFSF14 reduced EPC proliferation and endothelial tube formation. 32In C57BL/6 mice with streptozotocin induced diabetes and HLI, upregulating lysine demethylase 4B activated the canonical wingless (Wnt/β-catenin) pathway, which was associated with upregulation in SDF-1ά and vascular endothelial growth factor ά (VEGF).These effects significantly improved blood flow recovery compared to control mice. 35A number of studies have also implicated microRNAs (miRs) in controlling bone marrow cell contribution to angiogenesis and arteriogenesis. 36,37Cheng and colleagues studied miRs in patients with PAD and mice models of acute HLI secondary to Femoral artery ligation (FAL) and more gradual HLI secondary to placement of ameroid constrictors around the femoral artery (Supplement Figure 2). 36,37They identified 22 miRs including three of the four members of the miR-181 family (miR-181a-5p, miR-181b-5p and miR-181c-5p) that were downregulated in the plasma of both patients with diabetes and PAD, and mice with HLI. 37These miR-181 members were also downregulated within the ischaemic limbs of mice with impaired glucose tolerance compared to those with normal glucose handling. 37C57BL/6 mice with global deficiency in alleles miR-181a2b2 had significantly worse recovery of blood supply following HLI induction compared to wild type controls. 37Bone marrow transplant and cell specific transgenic studies suggested that miR-181 deficiency impaired Ly6Chi monocyte mobilisation from the bone marrow without local effects in endothelial cells.The impairment in blood flow recovery and the density of new capillaries in these mice could be rescued by infusion of Ly6Chi monocytes, illustrating the importance of these cells in angiogenesis.

| Local effects within the vasculature and ischaemic limb
The growth of new capillaries and remodelling of existing collaterals is also dependent on the function of endothelial cells and vascular smooth muscle cells within the lower limb vessels. 3,5Numerous novel local factors controlling angiogenesis and arteriogenesis have been identified in recent studies in mouse HLI models (Table 1).One of these factors is an apolipoprotein L domain containing 1 (Apold1), which is predominantly expressed in endothelial cells and has been shown to be upregulated in response to ischaemia.A recent study found that C57BL/6 mice deficient in Apold1 had significantly worse recovery of blood supply following HLI as a result of reduced endothelial cell proliferation and contribution to new blood vessel formation. 38miR-130b has been found to improve limb blood supply.
miR-130b was downregulated in the plasma of C57BL/6 mice and patients with limb ischaemia but upregulated in the ischaemic limb muscle compared to healthy tissue. 36In db/db (diabetic) mice, no upregulation of miR-130b in ischaemic leg muscle was identified.In MiRs have also been suggested to be responsible for the improved limb blood supply stimulated by bone marrow-derived mesenchymal stem cells. 39Extracellular vesicles derived from stem cells exposed to hypoxia significantly improved blood flow recovery in C57BL/6 mice.
This effect was associated with upregulation in miR-34c, M2 polarisation of macrophages and increased capillary density.The effects were partially blocked by deficiency in miR-34c, suggesting an important role in promoting angiogenesis.Other approaches of promoting M2 polarisation of macrophages have also been reported to promote blood flow recovery in C57BL/6 mice as discussed earlier. 20tric oxide (NO) release promotes angiogenesis and arteriogenesis. 40,41The glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide was reported to upregulate endothelial nitric oxide synthase (eNOS) in C57BL/6 mice with hyperglycemia and was shown to improve limb blood flow. 42Riociguat is a member of a novel class of soluble guanylate cyclase stimulators that increase NO release.Riociguat was reported to improve blood supply in C57BL/6 mice following HLI. 43Another soluble guanylate cyclase stimulator, praliciguat, has also been reported to improve limb blood supply in a mouse model of diabetes. 44Tang and colleagues found that levels of long noncoding RNA that enhances eNOS expression (LEENE) were decreased in the intima of mesenteric arteries from patients with diabetes compared to controls. 45In vitro LEENE increased expression of VEGF receptor 2 and eNOS in endothelial cells.C57BL/6 with LEENE deficiency had significantly impaired recovery of blood supply from FAL. 45 These findings suggest that LEENE expressing RNA could be a novel treatment for limb ischaemia.
Trimetazidine, a drug used to treat angina pectoris, inhibits 3ketoacyl-CoA thiolase favouring glucose rather than fatty acid oxidation, and is proposed to be cytoprotective during ischaemia.
Yang and colleagues found that trimetazidine improved semiquantitative scores for limb ischaemia and function in hyperglycaemic db/db mice with HLI, which was associated with increased capillary density. 46Analysis of endothelial cells from people with diabetes showed upregulation of the G protein-coupled receptor 39 (GPR39). 47In vitro GPR39 acted to impair endothelial cell proliferation, migration and tube formation through blocking the sonic hedgehog pathway by binding to a repressor called suppressor of fused homologue.In C57BL/6 mice with a diabetes phenotype, an adenovirus delivering mRNA overexpressing GPR39 improved hind limb blood supply recovery. 47

| TARGETS FOR TREATING LIMB ISCHEMIA FROM FINDINGS OF RECENT CLINICAL TRIALS
9][50][51][52][53][54][55][56][57][58][59] Women were under-represented, being between 0% and 38% of the enroled patients (Table 2), thus highlighting the unmet need of gender equality in this field.9][50][51][52][53][54][55][56][57] The VOYAGER sub study reported the efficacy of the anti-coagulant drug rivaroxaban at low dose in patients with PAD undergoing open surgical revascularisation, finding that the composite outcome of acute limb ischaemia, major amputation, myocardial infarction, ischaemic stroke and cardiovascular death were significantly reduced by allocation to rivaroxaban. 58Findings were similar in the COMPASS trial. 60Another trial tested a programme designed to improve patient self-management after major amputation for CLTI and found no effect of the intervention on musculoskeletal function but a significant improvement in the psycho-social and quality of life outcome measure. 599][50][51][52][53][54][55][56][57] Accepting this limitation, some promising results were reported for HGF and a number of cell therapies (Table 2).Gu and colleagues tested a HGF plasmid within two cohorts with ischaemic rest pain (n = 119) or ischaemic ulceration (n = 121) at doses ranging between 4 and 8 mg. 48The proportion of patients with diabetes was not reported.In the cohort with rest pain, HGF plasmid significantly improved the proportion of participants with complete resolution of pain (Table 2).In the cohort with ulceration, no significant effect on the primary outcome was noted.No safety concerns were reported.An earlier trial by the same investigators reported that HGF plasmid significantly reduced pain severity. 49In the highest dose HGF group (24 mg), there was also a significantly greater proportion of participants with complete ulcer healing in comparison to controls. 49Of note, the administration of the growth factor required up to 32 injections into the leg repeated on three separate days that may not be well-tolerated by patients.
Another open label randomised trial in 20 Italian participants with a diabetes-related foot ulcer and CLTI reported that a gel prepared from umbilical cord blood significantly improved percentage ulcer area reduction by 30 days. 51arma and colleagues reported on a placebo-controlled trial testing the effect of intra-arterial injections of bone marrow-derived stem cells. 50Approximately 69% of the 81 participants had CLTI and approximately one third had diabetes.The intervention doubled the proportion of participants with improvement in measures of leg blood supply, including an increase in ankle brachial index of ≥0.1 and in transcutaneous oxygen pressure of ≥15% (Table 1). 50There was a significant reduction in the rate of major amputation by comparison to controls.Liotta and colleagues reported on an open label randomised trial comparing administration of EPCs with non-enriched mononuclear cells in 40 participants with CLTI, of whom 17 had diabetes. 55The investigators reported that both groups had significant improvement from baseline in contrast ultrasound assessed microvascular circulatory flow, rest pain, ankle brachial index and transcutaneous oxygen pressure. 55The outcomes in the other cell therapy trials reported over the last 5 years have been less encouraging despite similar or larger sample sizes (Table 2).The reasons for this are unclear but could include heterogeneity in routes of administration, cell extraction methods, populations and outcome measures.
2][63] The HOPE CLTI is further testing the plasmid encoding HGF in two cohorts with rest pain (n = 150) and ischaemic ulceration (n = 240). 61The SAIL trial is testing bone marrow derived-mesenchymal stem cells in a small trial of patients with CLTI (n = 66). 63Finally, the GENEPAD trial is testing the value of CYP2C19 gene sequencing prior to the choice of anti-thrombotic therapy in patients with PAD (n = 2276).This open label trial is based on the prior finding that approximately 30% of patients are unable to adequately metabolise clopidogrel to the active antiplatelet form. 62It is hoped that these trials will shed additional light on the value of these therapies and approaches to managing people with CLTI.In addition, it is important that future trials focus on people with diabetes who have a higher risk of amputation and in whom specific pathways involved in improving blood supply to ischaemic tissues seem to be impaired, as discussed above.

GOLLEDGE ET AL.
T A B L E 2 Recent randomised controlled trials testing medical or rehabilitation therapies for peripheral artery disease (PAD) which included patients with chronic limb threatening ischaemia (CLTI).GOLLEDGE ET AL.
-9 of 12 T A B L E 3 Summary of targets identified in recent mouse models and clinical studies to upregulate or downregulate in order to improve limb blood supply.

F I G U R E 1
Recent findings of experimental studies on targets for novel treatments for ischaemic foot disease.Proposed mechanisms for angiogenesis and arteriogenesis and potential drug targets for medical revascularisation based on the findings of recent animal and clinical research.Beneficial and detrimental effects on revascularisation were denoted in purple and red colours respectively.Potential treatment strategies were presented in a golden yellow colour box.CD, Cluster of differentiation; eNOS, Endothelial nitric oxide synthase; G-CSF, Granulocyte colony stimulating factor; GPR39, G protein couple receptor 39; miR, MicroRNA; MMP, Matrix metalloproteinase; NET, Neutrophil extracellular traps; NO, Nitric oxide; oxLDL, Oxidised low density lipoprotein; PAD4, Peptidyl arginine deiminase 4; SDF1α, stromal cell-derived growth factor-1α; SHH, Sonic hedgehog; TGF, Transforming growth factor; TNF, Tumour necrosis factor; VEGFR, Vascular endothelial growth factor receptor; WNT, Wingless.T A B L E 1 Drug targets from recent rodent models of hind limb ischaemia (HLI).
vitro, D-glucose was downregulated, whereas VEGF and hypoxia upregulated miR-130b in human umbilical endothelial cells.Endothelial cells overexpressing miR-130b had enhanced proliferation and migration in vitro.Injection of miR-130b mimics into the gastrocnemius muscle of db/db mice significantly improved blood flow recovery and reduced necrosis score following FAL but not femoral artery ameroid constriction.The improved blood supply was accompanied by increased capillary density and reduced expression of TGF-β superfamily member inhibin-b-A (INHBA).Furthermore, silencer RNA targeting INHBA injected into the gastrocnemius muscle of db/db mice after FAL significantly improved blood flow recovery and necrosis score.This suggested that miR-130b overexpression and downregulation of INHBA are targets for promoting angiogenesis.

2 ,
trans-cutaneous oxygen pressure.a Mean visual analogue scale pain score and 95% confidence intervals.b Sub-study of the main trial focusing on patients having bypass surgery.c Include above knee, below knee and trans-metatarsal amputation for CLTI.d Compared with peripheral blood mononuclear cells in the control group.e Compared with bone marrow-derived mononuclear cells.f Including the control group which in the trials of HGF consisted of only one placebo group for the three different doses.The outcomes and numbers for the placebo group have been repeated as the comparator.g All part of the same trial and compared to the same one control group.h More patients originally randomised but only limited numbers reported.