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Review

Translational Relevance of Advanced Age and Atherosclerosis in Preclinical Trials of Biotherapies for Peripheral Artery Disease

by
Keith A. Webster
1,2
1
Vascular Biology Institute, University of Miami, Miami, FL 33146, USA
2
Department of Ophthalmology, Baylor College of Medicine, Houston, TX 77030, USA
Genes 2024, 15(1), 135; https://doi.org/10.3390/genes15010135
Submission received: 1 December 2023 / Revised: 8 January 2024 / Accepted: 17 January 2024 / Published: 22 January 2024
(This article belongs to the Special Issue Advances in Gene Therapy: Novel Vectors and Strategies)
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Abstract

:
Approximately 6% of adults worldwide suffer from peripheral artery disease (PAD), primarily caused by atherosclerosis of lower limb arteries. Despite optimal medical care and revascularization, many PAD patients remain symptomatic and progress to critical limb ischemia (CLI) and risk major amputation. Delivery of pro-angiogenic factors as proteins or DNA, stem, or progenitor cells confers vascular regeneration and functional recovery in animal models of CLI, but the effects are not well replicated in patients and no pro-angiogenic biopharmacological procedures are approved in the US, EU, or China. The reasons are unclear, but animal models that do not represent clinical PAD/CLI are implicated. Consequently, it is unclear whether the obstacles to clinical success lie in the toxic biochemical milieu of human CLI, or in procedures that were optimized on inappropriate models. The question is significant because the former case requires abandonment of current strategies, while the latter encourages continued optimization. These issues are discussed in the context of relevant preclinical and clinical data, and it is concluded that preclinical mouse models that include age and atherosclerosis as the only comorbidities that are consistently present and active in clinical trial patients are necessary to predict clinical success. Of the reviewed materials, no biopharmacological procedure that failed in clinical trials had been tested in animal models that included advanced age and atherosclerosis relevant to PAD/CLI.

1. Introduction

Peripheral artery disease (PAD), the third leading cause of atherosclerotic morbidity after coronary heart disease and stroke, refers primarily to lower limb ischemia and includes asymptomatic lower limb PAD, intermittent claudication (IC), and critical limb ischemia (CLI). PAD affects approximately 6% of adults globally and about 8.5 million people in the United States [1,2,3]. Incidence is markedly age-dependent, increasing from about 5% in subjects aged 40 to 44 years to >12% at age 70 to 74, with some studies reporting IC in up to 35% of patients over 50 years of age, and 1% to 2% with CLI [4,5,6]. Medical therapies to ameliorate hyperlipidemia, hypertension, and hyperglycemia combined with thrombolytics or fibrinolytics reduce morbidity and mortality related to cardiovascular events but have limited impact on PAD symptoms or disease progression [7,8]. Other major risk factors for PAD include smoking, sedentary lifestyle, high BMI, and elevated systemic inflammatory markers [4,9]. Currently, only supervised walking programs and the phosphodiesterase type 3 (PDE3) inhibitor cilostazol, an antiplatelet vasodilator, provide symptomatic benefits including improved pain-free walking time (PFWT). Surgical or endovascular revascularization remain primary interventions for lifestyle-limiting claudication and CLI, but this option is not available for up to 40% of CLI patients [10,11]. Despite two decades of intense basic and translational research efforts to develop gene and/or cell therapies, no new treatments have been approved in the US or EU, leading some to question whether PAD/CLI is even amenable to such biotherapies, especially gene therapy. Positive results of preclinical studies along with partial successes in some clinical trials of gene therapy, and broader successes of cell therapies, provide important information on the most promising pro-angiogenic biopharmacological strategies, but reveal larger protocol and translation flaws, and limited predictive value of preclinical models. This review discusses the failures of earlier gene therapy clinical trials, the possible reasons for such failures, impacts on ongoing biopharmacology research for PAD, and avenues to resolve the dilemmas of translation and optimization. The broad interpretation that gene therapy for PAD/CLI has failed should be tempered by the approvals and ongoing application of plasmid (p)VEGF and pHGF, respectively, in Russia and Japan, and the significant, albeit limited, efficacies seen for gene therapy on some clinical parameters such as rest pain and ulcer healing. Such spasmodic efficacy suggests inadequate strategies of implementation rather than fundamental misconceptions related to the approach and invites further analysis and optimization.

2. Literature Review

2.1. Meta-Analyses of Early Clinical Trials of Genes and Cells

Whereas early meta-analyses of gene and stem cell clinical trials of patients with PAD and CLI confirm universal safety of the procedures, efficacy to enhance blood flow, prevent or delay amputation and/or death, and improve PFWT and/or quality of life have been underwhelming. Genes tested in these trials include VEGF, FGF, HIF-1α, HGF, Del-1, SDF-1, and ZFP-VEGF (an engineered zinc finger transcription factor of VEGF-A), delivered by plasmid or adenoviral vectors (reviewed in: [7]). Cell therapies include bone marrow mononuclear cells (BMMNC), peripheral blood mononuclear cells (PBMNC), mesenchymal stem cells (MSC), endothelial progenitor cells (EPC), and smooth muscle cells (SMC). Figure 1 shows the genes and cells that have undergone positive preclinical evaluation and progressed to clinical trials by IA or IM delivery of recombinant human proteins, plasmids, or viral vectors. In the first meta-analysis of Phase 2 randomized, controlled clinical trials (RCT) of gene and cell therapies for PAD/CLI, De Haro et al. [12] reported that therapeutic angiogenesis significantly improved clinical outcomes including peak PFWT relative to placebo in both PAD and CLI cohorts. The authors concluded that gene and cell therapies were safe, well tolerated, and conferred significant efficacy for both PAD and CLI patients (see Table 1 and Refs. [13,14,15,16,17,18] therein). Four years later, using similar analyses that included updated trial results, Steiner and Hammer [19] concluded from 12 PAD/CLI gene therapy RCTs and a total of 1494 patients that endpoints were not significantly improved in the majority of studies. This meta-analysis showed neither significant benefit nor harm for gene therapy across all-cause mortality, amputations, or ulcer healing, and no differences in outcomes between patients with PAD or CLI. The meta-analysis included the Talisman 201 Phase II and TAMARIS Phase III RTCs of plasmid (p)FGF1 as well as pHGF, Ad- and pVEGF, pDel-1 and Ad-HIF1α, (see Table 2 and Refs. [20,21,22,23,24,25,26,27] therein); TAMARIS, with 525 CLI patients, was the largest worldwide gene therapy trial ever conducted [20,28]. Subsequent meta-analyses confirmed these results, including a study with 1988 PAD/CLI patients that reported no improvement of amputation-free survival, major amputation, or death by gene therapy relative to placebo [29]. These and other disappointing results that culminated with the withdrawal in 2016 of the multinational Phase III AGILITY RTC of pHGF for CLI (NCT02144610), brought this phase of translational research to an end and led to predictions that clinical trials of therapeutic angiogenesis by gene therapy for PAD were over [7].
By conferring more comprehensive stimuli involving multiple cytokines and growth factors with autocrine and paracrine angiogenic effects, cell therapy has been considered potentially superior, although more expensive and technically more demanding, than proteins or genes for promoting angiogenesis and tissue salvage in PAD. TACT (therapeutic angiogenesis using cell transplantation) was the first major cell clinical trial for PAD [18] that compared BMMNC with PBMNC. The study reported safety and significantly greater improvements in ABI, rest pain, and PFWT at 24 weeks in the BMMNC group. PROVASA (intra-arterial administration of BMMNC in patients with CLI), a Phase II RTC of intra-arterial BMMNC versus placebo, reported no difference in ABI or limb salvage but significant improvements in ulcer healing and rest pain [26]. The largest JUVENTAS (rejuvenation EPC via transcutaneous intra-arterial supplementation) trial of CLI patients with intra-arterial EPCs versus placebo, reported no significant differences in major amputation rate, quality of life, rest pain, ABI, or TcPO2 [27]. In a 2017 meta-analysis of autologous cell therapy that included 19 RCTs (837 patients), 7 nonrandomized trials (338 patients), and 41 noncontrolled studies (1177 patients), Rigato et al. [30] reported that cell therapy reduced the risk of amputation by 37%, and improved amputation-free survival and wound healing by 18% and 59%, respectively. Cell therapy also significantly increased ABI and TcPO2, and reduced rest pain. The authors noted that the efficacy of end points was no longer significant in placebo controlled RCTs and disappeared in RTCs with a low risk of bias. In a 2018 meta-analysis of RCTs of autologous stem cell therapy in CLI, Xie et al. [31] reported significantly improved ABI, TcO2, PFWT, as well as ulcer healing, reduced amputation rates, and increased angiogenic scores in the cell therapy group relative to controls. The analysis concluded that while cell therapy may be safe and effective, higher quality and larger RCTs are still required to support clinical application. In another recent (2019) meta-analysis of autologous stem cell therapy [32] that included 27 RCTs and 1186 patients, Gao et al. reported significantly improved healing of ulcers, ABI, TcO2, and PFWT, reduction of rest pain, and minor amputation rates, but no significant improvement in major limb salvage. The authors acknowledged high risk of bias and low-quality of evidence outcomes and concluded that autologous stem cell therapy may have a positive effect on “no-option” patients with PAD but did not significantly impact major limb amputation. In a meta-analysis of autologous cell therapy for CLI including 12 RCTs and 630 patients, Pu et al. [33] reported significantly improved total and major amputation rates, ABI, TcO2, and rest pain score compared with placebo or standard care but no change in all-cause death or ulcer size and concluded that autologous cell therapy conferred benefit to CLI patients in terms of limb salvage, perfusion, and rest pain alleviation. The results of the meta-analyses of autologous cells are summarized in Table 3.
In their review of angiogenic cell therapy for CLI up to and including 2021 data, Beltrán-Camacho et al. [34], concluded that 20 years of clinical trials with autologous stem cells confirms safety and promising efficacy but due to high variability between studies and low to moderate quality of data, it remains unclear which cells, doses, or routes of administration are optimal. One possible problem cited by several authors is that most studies are supported by sponsors with potentially conflicted interests and reporting is often incomplete and/or subjective. However, despite this, most authors agree on the safety and feasibility of cell therapies and concur that they represent a promising approach for no option CLI, a population that represents 50% of CLI patients [35,36]. Ongoing trials are also testing secreted products such as exosomes that avoid the potential hazards and regulatory/technical demands of live autologous cell therapy and optimizing cells by preconditioning and/or genetic engineering [37,38,39]. Although, autologous cell-based approaches now appear to show greater promise than gene therapy, the procedures are more expensive, labor intensive, and technically demanding.

2.2. Recent and Ongoing Clinical Trials of Genes and Cells II

With the exception of ongoing trials of pVM202 and JVS-100 (see below), the 2017 prediction by Iyler and Annex [7] that “termination of the multinational Phase III AGILITY trial may well end gene therapy trials of therapeutic angiogenesis for peripheral arterial disease”, appears to have been substantially validated within the US. Despite insufficient evidence of efficacy for approval in the US, pVEGF-165 (Neovasculgen) was approved to treat CLI patients by the Russian Ministry of Healthcare in 2011 and the Ukraine equivalent in 2013. The decisions were based on positive safety/efficacy findings of pre-clinical studies and a Phase IIB/III clinical trial [40,41,42]. Similarly, pHGF (Collategene) was approved by the Japanese Ministry of Health to treat ulcers in no-option CLI patients in 2019. This approval was based on positive results from preclinical and clinical studies including a Phase III RCT [24,43,44,45,46]. Clinical studies of pHGF containing two isoforms of human pHGF (HGF728 and HGF723) to treat PAD are also ongoing in the US and China. HI-PAD, a Phase II RTC of pVM202 in the US was initiated in 2018 (NCT03363165) and a Phase III trial of NL003 (HGF-X7) in China was initiated in 2019 (NCT04274049). Awaited outcome results include wound healing, rest pain, and limb salvage. In a related Phase III RCT of patients with painful diabetic peripheral neuropathy, Kessler et al. [47] recently reported significant alleviation of pain by pVM202 in one arm of the trial. A Phase II RCT in Poland assessed safety and efficacy of a bicistronic plasmid vector expressing human VEGF165 and HGF (pIRES/VEGF165/HGF) by intramuscular injections in diabetic CLI patients [48]. Preliminary results indicate safety and efficacy with significantly increased ABI, reduced rest pain, and improved vascularization assessed by computed tomography angiography. In Beijing, China, a dose escalation, safety, and tolerability Phase I clinical trial of human FGF-2 delivered via intramuscular injection of Sendai virus (SeV-hFGF2) to CLI patients, initiated in 2018, is underway (NCT03668353). In another Phase II RCT, Shishehbor et al. reported that gene therapy with JVS-100 (pSDF-1) conferred no significant improvement in outcomes of patients undergoing revascularization [49]. The Libella gene therapy trial in Colombia is an ongoing Phase I safety and tolerability trial of intravenous adeno-associated virus (AAV) expressing the human telomerase reverse transcriptase (AAV-hTERT) in CLI patients (NCT04110964). Telomerases protect against age- and cell division-dependent telomere shortening, thereby delaying senescence and preserving gene function. hTERT was shown to augment VEGF-A activity and enhance the regenerative properties of endothelial progenitor cells (EPC) in vitro and in vivo. Adenovirus (Ad)-hTERT combined with Ad-VEGF was shown to enhance angiogenesis, vascular remodeling, and perhaps muscle regeneration in a rat CLI model [50], and the hope is that AAV-hTERT ameliorates telomere dysfunction and augments vascular regeneration in elderly PAD patients. A parallel trial will test AAV-hTERT in Alzheimer’s patients. Recruitment for the Libella trials commenced in 2019.

2.3. Predictive Value of Preclinical Models

Despite the Russian and Japanese approvals, respectively, of pVEGF and pHGF, and the ongoing trials of pVM202 and JVS-100, the universal failure of large Phase III US-based clinical trials of gene therapy for CLI has markedly dampened enthusiasm for the technology and prompted speculation on the reasons for the failures [34,51,52]. Possible explanations include: (1) use of single angiogenic growth factors that generate immature leaky capillaries reminiscent of pathological angiogenesis [53]; (2) limitation of most interventions to stimulate only angiogenesis when revascularization may require enhanced arteriogenesis and vasculogenesis; (3) insufficient level, duration, targeting, and regulation of transgene expression; and (4) inadequate predictive value of preclinical models that do not include comorbidities of PAD/CLI patients [1,2,3,54,55]. Plasmids have been the vectors of choice for all major trials so far, whereas AAV and lentiviral vectors may provide higher levels of sustained, regulable expression [56]. However, the inadequacy of preclinical models to accurately predict clinical translation stands out as a major reason for the failed gene therapy trials. Two properties of the most used mouse models that fail to replicate clinical targets include: (1) acute ischemia by femoral artery ligation and excision (FAL) that does not replicate occlusion by progressive atherosclerotic narrowing by arterial plaque, and (2) absence of comorbidities that determine the responsiveness of ischemic limb tissues to pro-angiogenic stimuli. Comorbidities of PAD/CLI patients that are rarely incorporated into mouse models include age, atherosclerosis, hypertension, hyperglycemia, hyperlipidemia, diabetes, smoking, sedentary lifestyle, and elevated systemic inflammation [51,57,58,59]. Of these, advanced age and severe atherosclerosis are the only comorbidities that are consistently present in patients with clinical PAD/CLI. Other comorbidities that exacerbate clinical PAD and are variously present in clinical trial subjects are usually well-controlled by standard pharmacology that includes antithrombotic drugs, and medications to treat dyslipidemia, hypertension, and diabetes. Such management reduces the risk of major adverse events (AMI and stroke) but does not alter the course of PAD progression or the outcome of biopharmacological (gene/cell) clinical trials [1,3]. The average age of patients in the major Phase III clinical trials of gene and cell therapies for CLI is >70 years and all patients were symptomatic with leg pain and ulcers secondary to chronic ischemia caused by atherosclerotic plaque build-up in the in-flow blood vessels [60]. For example, the mean age of patients in the TAMARIS trial was 70 years (equivalent to 25-month C57BL/6 mice), wherein atherogenic stenoses were present in >95% of infrainguinal arteries, 66% of thigh arteries, and 94% of arteries below the knee. In total, 70% of patients had more than one diseased artery, 80% were hypertensive, 60% hypercholesterolemia, 61% former or current smokers, 53% diabetic, and 18% obese. Similar hemodynamic and angiographic patterns were typically reported in all regional areas, as well as in diabetic and non-diabetic patients. Therefore, the vasculatures of aged CLI patients with chronic, severe atherosclerosis are unlikely to replicate the responses to angiogenic stimulation of young healthy mice with acute ischemic damage.

2.4. Age and Atherosclerosis in Mouse CLI Models

Multiple hindlimb FAL models have been described wherein the intrinsic aptitude for vascular regeneration and functional recovery is dependent on the mouse strain [57,61,62,63,64]. Whereas most models are made acutely ischemic via double FAL with FA excision, new models more accurately replicate occlusion in clinical PAD [58,65,66]. Strain- and age-dependent responses to angiogenic stimulation, including roles of circulating PBMNC and BMMNC, have been described [67,68]. In the latter study, Bosch-Marce et al. compared intrinsic perfusion recovery rates of young (2 months), mid-aged (8 months), and old-aged (20 months) C57BL/6 mice after FAL. Old-age mice salvaged only 40% of limbs and achieved < 30% perfusion compared with young mice. The authors attributed the differences to loss of mobilization of VEGFR2+/CD34+ angiogenic cells and deceased levels of pro-angiogenic cytokines after FAL in the ischemic limbs of aged mice. Reductions of cytokines included ANGPT1 & 2 (<10%), HIF-1α (<25%), MCP-1(<10%), PLGF (<10%), SCF (<10%), and SDF-1 (~10%). These trends were confirmed in other studies that reported similar ischemia, but <50% recovery of limb function in 18-month versus 3-month-old mice after FAL, and diminished arteriogenesis in the ischemic hind limbs of older mice [64,69]. Collaterals in aged mice are less able to remodel and enlarge in response to FAL because of deficient eNOS production and increased susceptibility of ECs and SMCs to apoptosis [70]. HIF-1α levels are decreased in the ischemic hind limbs of aged mice and correlate with reduced angiogenic factors, lower recruitment of angiogenic cells, and loss of perfusion recovery after FAL [70]. Deficiency of Klotho, an anti-aging gene in mice confers a phenotype equivalent to human aging that includes short lifespan, stunted growth, vascular calcification, and atherosclerosis [71,72,73]. Mice with heterozygous deficiency of the klotho gene show impaired neovascularization and perfusion recovery after FAL with markedly decreased nitric oxide release and reduction of BMMNCs [74].
Atherosclerosis progression involves the accumulation of lipids, inflammatory cells, and smooth muscle cells in arterial walls that culminate in necrosis, fibrosis, and calcification [75]. Systemic inflammation is associated with severe PAD and atherothrombotic narrowing during PAD promotes changes in the circulatory system and tissues including NO-dependent compensatory responses, angiogenesis and arteriogenesis, and detrimental effects including microvascular dysfunction, myopathy, fibrosis, and tissue necrosis [76,77]. PAD is associated with reduced calf skeletal muscle area and density, increased calf muscle fat infiltration, increased oxidative stress, impaired mitochondrial activity, and smaller myofibers [78,79]. ApoE-knockout (ApoE-/-) mice mimic traits of human atherosclerosis including inflammation and metabolism and are commonly used to simulate the effects of atherosclerosis and dyslipidemia [80,81]. Studies from multiple groups, including the author’s, have demonstrated that the evolution of atherosclerosis in ApoE-/- mice is paralleled by infiltration of inflammatory cells and progressive loss of mobility of BMMNCs with reduced levels of angiogenic factors including SDF-1 [82,83,84,85]. ApoE-/- C57BL/6 mice develop significant atherosclerotic lesions in the ascending aorta, carotid, femoral, and popliteal arteries [86,87,88], coincident with delayed recovery from ischemia and stunted response to angiogenic therapy [89,90]. Couffinhal et al. [91] first showed that recovery of young C57BL/6 ApoE-/- mice from hindlimb FAL was markedly attenuated relative to WT controls. Capillary density, CD-31, and VEGF that were significantly reduced in the ApoE-/- group paralleled increased infiltration of inflammatory cells, leukocytes, macrophages, and T-lymphocytes. Similar results were reported by Xie et al., effects that were partially reversed by delivery of ZFP-32E, a zinc finger DNA-binding transcription factor of VEGF [92]. Using a FAL model that included Western diet and 7-month aged ApoE-/- mice, Lejay et al. [93] reported more severe mitochondrial dysfunction and increased oxidative stress after FAL of ApoE-/- mice relative to control WT mice. Blunted responses of ApoE-/- mice to angiogenic stimuli have been variously attributed to dysregulated expression of miRs that regulate angiogenesis and vasculogenesis in PAD [94,95,96,97,98,99]. Peck et al. [61] subjected 8-month-old ApoE-/- fed a normal diet to exercise training after FAL and documented responses that mimic CLI patients. They proposed that such aged ApoE-/- mice represent a more appropriate hindlimb ischemia model to accurately evaluate therapeutic strategies for human PAD/CLI. By regulating inflammation and EC angiogenesis, respectively, miR-146b and miRNA-27b are implicated in the suppressed angiogenic responses of ApoE-/- mice [100,101]. Together, results from multiple sources concur that age and atherosclerosis create toxic environments for vascular regeneration, and their absence represent a shortcoming of therapeutic angiogenesis preclinical trials to date. By supporting optimization on a relevant background, inclusion of age and atherosclerosis is expected to provide more accurate predictions of clinical success.

2.5. Recent and Ongoing Preclinical Trials of Genes, MiRs, and NO-Donors

AGGF1 (angiogenic factor with G-patch and Forkhead-associated domain-1) binds the integrin α5β1 receptor on ECs [102] and is the earliest known regulator of multipotent hemangioblast specification, regulating hematopoiesis and differentiation of endothelial lineages [103]. AGGF1 regulates EC proliferation, adhesion, migration, and capillary tube formation [104,105] and promotes angiogenesis as potently as VEGF-A [106]. In hindlimb ischemia mouse models, pAGGF1 promoted therapeutic angiogenesis more efficiently than FGF2 [107,108]. Transplantation of AGGF1-transduced EPCs conferred limb salvage, reperfusion, and exercise tolerance in high-fat diet and db/db diabetic mouse hindlimb ischemia models [109]. AGGF1 also binds and regulates VSMC phenotypic switching, proliferation, and migration [110], and drives therapeutic angiogenesis through a pathway of integrin α5β1, FAK, Src, and AKT signaling. The authors predict that AGGF1, through its roles in regulating vasculogenesis, angiogenesis, and vascular development, represents a promising target for clinical development to provide a more effective therapy for PAD/CLI [111].
Anti-angiogenic VEGF165b is an alternative spliced isoform of VEGF-A that is increased in ischemic muscle [2] and competes with pro-angiogenic isoforms of VEGF-A for binding to VEGFR2 [112]. VEGF165b is a weak agonist of VEGFR2, and the interaction lacks the downstream signaling required for an angiogenic response, making it a competitive inhibitor of the VEGFR2-AKT-ERK-eNOS-NO angiogenic pathway [113,114,115]. By suppressing NO production, elevated VEGF165b in muscles of PAD/CLI patients may contribute to the absence of clinical benefit seen in VEGF-A clinical trials or of NO supplementation by L-arginine in PAD patients. With the rationale that PAD/CLI patients have chronically reduced responses to NO signaling and thence therapeutic angiogenesis, Kuppuswamy et al. [2] recently showed that delivery of an anti-VEGF165b antibody significantly enhanced perfusion and increased microvascular density in three mouse PAD models with suppressed NO production including T2D and eNOS knock-out mice. The authors conclude that VEGF165b is a potential therapeutic target for patients with PAD where the VEGFR2-eNOS-NO pathway is impaired. The same group recently identified the IL-21 receptor (IL-21R) as causally linked to the differential responses of C57BL/6 versus Balb/c mice to hindlimb ischemia. High IL-21R expression in ECs of ischemic hindlimbs in C57BL/6 mice coincided with enhanced perfusion recovery whereas low IL-21R expression in Balb/C correlated with sustained perfusion deficit and greater tissue loss during HLI [61]. Genetic support linking the IL-21R with human PAD prompted the authors to target IL-21R for nitric oxide-independent angiogenesis in PAD [1].
MiR-15 and -16 belong to an extended miR-16 family that bind to Tie2 mRNA coding sequences (CDSs) and regulate angiogenesis by targeting VEGFR2 and FGFR1 [116]. The miRs are conserved between humans and mice [117]. MiR-15a and -16 are increased in serum and circulating proangiogenic cells (PACs) of CLI patients wherein serum concentrations predict amputation at 1-year post-revascularization [118]. Ex vivo transfection with miR-15a/16 inhibitors increase the potential of human PACs to induce therapeutic angiogenesis in mouse PAD models [118] and therapeutic angiogenesis is impaired in mice with miR-15a gene knock-in [119]. Local adenoviral delivery of a 15a/16 decoy increased Tie2 levels in ischemic skeletal muscle, improved perfusion recovery, and reduced toe necrosis. The results support further development of Ad-Decoy-15a/16 to treat human PAD/CLI.
MiR-150. Using next-generation sequencing and quantitative reverse transcription polymerase chain reaction analyses. Desjarlais et al. [120] reported that decreased levels of the proangiogenic microRNA miR-150 in ApoE-/- mice conferred decreased Src, eNOS, and Akt activities that was mechanistically associated with inefficient neovascularization following FAL. The effects were normalized by forced expression of an miR-150 mimic that the authors propose to represent a novel therapeutic strategy to improve ischemia-induced neovascularization in atherosclerotic conditions. However, this model does not represent the average age or severity of atherosclerosis of human CLI.
AAV-PFKFB3 delivery was shown to salvage limbs, increase perfusion, and improve muscle contractile function following FAL in BALB/c mice [121]. The model mimics CLI patients wherein compromised mitochondria and inflexible metabolism exacerbate myopathy. The authors report that the glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) was markedly induced in transgenic mice with defective mitochondrial metabolism caused by accumulated mutations in mtDNA, and this conferred resistance to ischemic myopathy by enhancing glycolysis and maintaining ATP in ischemic muscle. Muscles from CLI patients were shown to contain lower PFKFB3 relative to normal or claudication muscles and decreased glycolytic flux capacity. The results support reduced glycolytic flux as a common characteristic of failing CLI patient limb skeletal muscle that may be responsive to gene therapy with AAV-PFKFB3.
MPC-1011, an NO-donor, stimulates angiogenesis and arteriogenesis and improves hindlimb ischemia via a cGMP-dependent pathway involving VEGF and SDF-1α [122]. Atherosclerosis-impaired NO production and associated vascular dysfunction is well-documented in PAD patients and animal models [123,124,125,126,127,128]. Preclinical studies and small clinical trials confirm increased PFWT in PAD patients via NO augmentation with beetroot juice, derivates of dark chocolate, MitoQ, a mitochondria-targeted antioxidant, and PDE-V inhibitor sildenafil [129,130,131,132,133].
ACAT 1/2 inhibitors. High-intensity statins are recommended for patients with peripheral artery disease (PAD) and meta-analyses have shown that CLI patients benefit from statin therapy with significantly lower amputation rates (~25%) and fewer fatal events compared with control groups without statin therapy [134,135]. Attempts to augment this effect include inhibition of acyl-coenzyme A:cholesterol acyltransferase (ACAT; EC 2.3.1.26) enzymes that regulate cholesterol homeostasis by esterifying the 3-hydroxyl position of cellular free cholesterol with a fatty acid-CoA, creating cholesteryl ester (CE). ACAT1 is responsible for CE accumulation in macrophage foam cells and its inhibition was predicted to reduce cholesterol accumulation in atherosclerotic lesions. Positive anti-atherosclerosis activity of ACAT1 inhibitors in mouse and rabbit models supported three multi-center placebo controlled RCTs of two different ACAT1 inhibitors. However, human trials reported no significant improvement in atheroma volume regression, and in some cases, significantly increased major cardiovascular events in treated vs. placebo [136,137]. The reasons for the absence of clinical success are unclear, but there were indications of adverse effects of ACAT inhibition in some animal studies involving mitochondrial dysfunction, cytotoxicity, proinflammatory effects, and apoptosis, that the authors suggest may be alleviated by switching to ACAT2 inhibitors [137].
NLRP3 inflammasome stimulation by cellular stress activates caspase-1 and cleavage of pro-inflammatory cytokines IL-1β and IL-18, that trigger an inflammatory response. Quantitative trait locus mapping and molecular technologies in mice have identified new genetic loci (Ath28, Ath22, Ath26) and associated genes (Soat1, Gpnmb, AKR Pycard) associated with NLRP3 inflammasomes that determine macrophage phenotypes and associated atherosclerosis plaque and pathology [138,139,140]. Targeted inhibition of the NLRP3 inflammasome pathway represents another ongoing promising approach to slow atherosclerosis.

3. Summary and Conclusions

While the positive albeit variable results of cell therapies for PAD/CLI encourage further testing to optimize and consolidate cell types and procedures, the limited efficacies revealed by large clinical trials of gene therapy have led to reduced enthusiasm for continued development of this approach. However, gene therapy clinical trials were based on preclinical models that lacked predictive value for clinical translation, and much evidence suggests that more predictive models would identify different protocols with increased likelihood of clinical success. At minimum, such models would preclude expensive, time, and labor-intensive clinical development of products that were predestined to fail. Importantly, all Phase III gene therapy trials were limited to plasmid gene delivery, whereas more efficient viral delivery (AAV/Lentivirus) allows higher expression, targeting, and defined duration of transgene expression, as demonstrated in a PAD model and previously reviewed by the author’s group [56,141,142] and in other indications [143,144,145]. Newer gene therapy approaches, including those listed above, may benefit from preliminary testing in preclinical models that include advanced age and atherosclerosis (ApoE-/- mice) prior to clinical development. Such an application, while arduous, is not as labor intensive, time consuming, and expensive as the build-up and implementation of clinical trials; by some estimates the cost of TAMARIS was >$25M. While FDA IND approvals for cardiovascular indications require two animal species, relevant co-morbidities are not required and not routinely included.
Advanced age and atherosclerosis create toxic environments that render host tissues, vascular beds, and resident cells resistant to vascular regeneration and tissue salvage. Heightened local and systemic inflammation, suppressed expression of angiogenic and arteriogenic growth factors and cytokines, impaired NO production, dysregulated miRs that drive angiogenesis and arteriogenesis, microvascular dysfunction, increased oxidative stress, and impaired metabolic regulation including mitochondrial and glycolytic dysfunctions may contribute to muted responses to vasculogenesis. The effects worsen in parallel with age and severity of atherosclerosis. CLI patients in the major failed clinical trials of gene therapy were of advanced age (mean of 70 years) with severe atherosclerosis. To our knowledge, none of the angiogenic genes subject to clinical trial were tested in preclinical models that included equivalent backgrounds of advanced age and atherosclerosis. Mouse PAD/CLI models that include age and/or atherosclerosis show markedly reduced responses to both gene and cell treatments. Application of more inclusive models, including surgical techniques that more closely mimic the target population of PAD patients, is predicted to provide much needed information to optimize both gene and cell therapies for clinical application.

Funding

Supported by NIH grants # 5R24EY028764 and R01EY033805.

Conflicts of Interest

The author declares no conflicts of interest in the subject matter of the review.

Abbreviations

Peripheral artery disease: PAD; critical limb ischemia: CLI; pain-free walking time: PFWT; plasmid: p; vascular endothelial growth factor: VEGF; fibroblast growth factor: FGF; hepatocyte growth factor: HGF; hypoxia-inducible factor-1-alpha: HIF-1α; developmental endothelial locus-1: Del-1, stromal-derived factor 1: SDF-1; bone marrow mononuclear cells: BMMNC; peripheral blood mononuclear cells: PBMNC; mesenchymal stem cells: MSC; endothelial progenitor cells: EPC; smooth muscle cells: SMC; intra-arterial: IA; intramuscular: IM; quality of life: QoL; randomized, controlled clinical trials: RCT; ankle brachial ratio: ABI, total carbon dioxide:TcO2; adeno-associated virus: AAV; adenovirus: Ad; femoral artery ligation: FAL; angiopoietins: ANGPT; monocyte chemoattractant protein-1: MCP-1; placental growth factor: PLGF; stem cell factor: SCF; nitric oxide: NO.

References

  1. Annex, B.H.; Cooke, J.P. New Directions in Therapeutic Angiogenesis and Arteriogenesis in Peripheral Arterial Disease. Circ. Res. 2021, 128, 1944–1957. [Google Scholar] [CrossRef] [PubMed]
  2. Kuppuswamy, S.; Annex, B.H.; Ganta, V.C. Targeting Anti-Angiogenic VEGF165b-VEGFR1 Signalling Promotes Nitric Oxide Independent Therapeutic Angiogenesis in Preclinical Peripheral Artery Disease Models. Cells 2022, 11, 2676. [Google Scholar] [CrossRef] [PubMed]
  3. Ganta, V.C.; Annex, B.H. Peripheral vascular disease: Preclinical models and emerging therapeutic targeting of the vascular endothelial growth factor ligand-receptor system. Expert Opin. Ther. Targets 2021, 25, 381–391. [Google Scholar] [CrossRef] [PubMed]
  4. Fowkes, F.G.; Rudan, D.; Rudan, I.; Aboyans, V.; Denenberg, J.O.; McDermott, M.M.; Norman, P.E.; Sampson, U.K.; Williams, L.J.; Mensah, G.A.; et al. Comparison of global estimates of prevalence and risk factors for peripheral artery disease in 2000 and 2010: A systematic review and analysis. Lancet 2013, 382, 1329–1340. [Google Scholar] [CrossRef] [PubMed]
  5. Rooke, T.W.; Hirsch, A.T.; Misra, S.; Sidawy, A.N.; Beckman, J.A.; Findeiss, L.; Golzarian, J.; Gornik, H.L.; Jaff, M.R.; Moneta, G.L.; et al. A report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. J. Am. Coll. Cardiol. 2013, 61, 1555–1570. [Google Scholar] [PubMed]
  6. Hirsch, A.T.; Haskal, Z.J.; Hertzer, N.R.; Bakal, C.W.; Creager, M.A.; Halperin, J.L.; Hiratzka, L.F.; Murphy, W.R.; Olin, J.W.; Puschett, J.B.; et al. Guidelines for the Management of Patients with Peripheral Arterial Disease. Circulation 2006, 113, 463–654. [Google Scholar]
  7. Iyer, S.R.; Annex, B.H. Therapeutic Angiogenesis for Peripheral Artery Disease: Lessons Learned in Translational Science. JACC Basic Transl. Sci. 2017, 2, 503–512. [Google Scholar] [CrossRef]
  8. Mohler, E.R., 3rd; Hiatt, W.R.; Creager, M.A. Cholesterol reduction with atorvastatin improves walking distance in patients with peripheral arterial disease. Circulation 2003, 108, 1481–1486. [Google Scholar] [CrossRef]
  9. Criqui, M.H.; Aboyans, V. Epidemiology of peripheral artery disease. Circ. Res. 2015, 116, 1509–1526. [Google Scholar] [CrossRef] [PubMed]
  10. Norgren, L.; Hiatt, W.R.; Dormandy, J.A.; Nehler, M.R.; Harris, K.A.; Fowkes, F.G.; TASC II Working Group. Inter-Society Consensus for the Management of Peripheral Arterial Disease (TASC II). J. Vasc. Surg. 2007, 45, S5–S67. [Google Scholar] [CrossRef]
  11. Lawall, H.; Bramlage, P.; Amann, B. Stem cell and progenitor cell therapy in peripheral artery disease. A critical appraisal. Thromb. Haemost. 2010, 103, 696–709. [Google Scholar] [PubMed]
  12. De Haro, J.; Acin, F.; Lopez-Quintana, A.; Florez, A.; Martinez-Aguilar, E.; Varela, C. Meta-analysis of randomized, controlled clinical trials in angiogenesis: Gene and cell therapy in peripheral arterial disease. Heart Vessels 2009, 24, 321–328. [Google Scholar] [CrossRef] [PubMed]
  13. Lazarous, D.F.; Unger, E.F.; Epstein, S.E.; Stine, A.; Arevalo, J.L.; Chew, E.Y.; Quyyumi, A.A. Basic fibroblast growth factor in patients with intermittent claudication. J. Am. Coll. Cardiol. 2000, 36, 1239–1244. [Google Scholar] [CrossRef] [PubMed]
  14. Rajagopalan, S.; Mohler, E.R., 3rd; Lederman, R.J.; Mendelsohn, F.O.; Saucedo, J.F.; Goldman, C.K.; Blebea, J.; Macko, J.; Kessler, P.D.; Rasmussen, H.S.; et al. Regional angiogenesis with vascular endothelial growth factor in peripheral arterial disease: A phase II randomized, double-blind, controlled study of adenoviral delivery of vascular endothelial growth factor 121 in patients with disabling intermittent claudication. Circulation 2003, 108, 1933–1938. [Google Scholar] [PubMed]
  15. Lederman, R.J.; Mendelsohn, F.O.; Anderson, R.D.; Saucedo, J.F.; Tenaglia, A.N.; Hermiller, J.B.; Hillegass, W.B.; Rocha-Singh, K.; Moon, T.E.; Whitehouse, M.J.; et al. Therapeutic angiogenesis with recombinant fibroblast growth factor-2 for intermittent claudication (the TRAFFIC study): A randomised trial. Lancet 2002, 359, 2053–2058. [Google Scholar] [CrossRef] [PubMed]
  16. Mäkinen, K.; Manninen, H.; Hedman, M.; Matsi, P.; Mussalo, H.; Alhava, E.; Ylä-Herttuala, S. Increased vascularity detected by digital subtraction angiography after VEGF gene transfer to human lower limb artery: A randomized, placebo-controlled, double-blinded phase II study. Mol. Ther. 2002, 6, 127–133. [Google Scholar] [CrossRef] [PubMed]
  17. Grossman, P.M.; Mendelsohn, F.; Henry, T.D.; Hermiller, J.B.; Litt, M.; Saucedo, J.F.; Weiss, R.J.; Kandzari, D.E.; Kleiman, N.; Anderson, R.D.; et al. Results from a phase II multicenter, double-blind placebo-controlled study of Del-1 (VLTS-589) for intermittent claudication in subjects with peripheral arterial disease. Am. Heart J. 2007, 153, 874–880. [Google Scholar] [CrossRef]
  18. Tateishi-Yuyama, E.; Matsubara, H.; Murohara, T.; Ikeda, U.; Shintani, S.; Masaki, H.; Amano, K.; Kishimoto, Y.; Yoshimoto, K.; Akashi, H.; et al. Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: A pilot study and a randomised controlled trial. Lancet 2002, 360, 427–435. [Google Scholar] [CrossRef]
  19. Hammer, A.; Steiner, S. Gene therapy for therapeutic angiogenesis in peripheral arterial disease—A systematic review and meta-analysis of randomized, controlled trials. Vasa 2013, 42, 331–339. [Google Scholar] [CrossRef] [PubMed]
  20. Belch, J.; Hiatt, W.R.; Baumgartner, I.; Driver, I.V.; Nikol, S.; Norgren, L.; Van Belle, E. Effect of fibroblast growth factor NV1FGF on amputation and death: A randomised placebo-controlled trial of gene therapy in critical limb ischaemia. Lancet 2011, 377, 1929–1937. [Google Scholar] [CrossRef]
  21. Duong Van Huyen, J.P.; Smadja, D.M.; Bruneval, P.; Gaussem, P.; Dal-Cortivo, L.; Julia, P.; Fiessinger, J.N.; Cavazzana-Calvo, M.; Aiach, M.; Emmerich, J. Bone marrow-derived mononuclear cell therapy induces distal angiogenesis after local injection in critical leg ischemia. Mod. Pathol. 2008, 21, 837–846. [Google Scholar] [CrossRef] [PubMed]
  22. Powell, R.J.; Simons, M.; Mendelsohn, F.O.; Daniel, G.; Henry, T.D.; Koga, M.; Morishita, R.; Annex, B.H. Results of a double-blind, placebo-controlled study to assess the safety of intramuscular injection of hepatocyte growth factor plasmid to improve limb perfusion in patients with critical limb ischemia. Circulation 2008, 118, 58–65. [Google Scholar] [CrossRef] [PubMed]
  23. Nikol, S.; Baumgartner, I.; Van Belle, E.; Diehm, C.; Visoná, A.; Capogrossi, M.C.; Ferreira-Maldent, N.; Gallino, A.; Wyatt, M.G.; Wijesinghe, L.D.; et al. Therapeutic angiogenesis with intramuscular NV1FGF improves amputation-free survival in patients with critical limb ischemia. Mol. Ther. 2008, 16, 972–978. [Google Scholar] [CrossRef] [PubMed]
  24. Shigematsu, H.; Yasuda, K.; Iwai, T.; Sasajima, T.; Ishimaru, S.; Ohashi, Y.; Yamaguchi, T.; Ogihara, T.; Morishita, R. Randomized, double-blind, placebo-controlled clinical trial of hepatocyte growth factor plasmid for critical limb ischemia. Gene Ther. 2010, 17, 1152–1161. [Google Scholar] [CrossRef] [PubMed]
  25. Creager, M.A.; Olin, J.W.; Belch, J.J.; Moneta, G.L.; Henry, T.D.; Rajagopalan, S.; Annex, B.H.; Hiatt, W.R. Effect of hypoxia-inducible factor-1alpha gene therapy on walking performance in patients with intermittent claudication. Circulation 2011, 124, 1765–1773. [Google Scholar] [CrossRef] [PubMed]
  26. Walter, D.H.; Krankenberg, H.; Balzer, J.O.; Kalka, C.; Baumgartner, I.; Schlüter, M.; Tonn, T.; Seeger, F.; Dimmeler, S.; Lindhoff-Last, E.; et al. Intraarterial administration of bone marrow mononuclear cells in patients with critical limb ischemia: A randomized-start, placebo-controlled pilot trial (PROVASA). Circ. Cardiovasc. Interv. 2011, 4, 26–37. [Google Scholar] [CrossRef] [PubMed]
  27. Teraa, M.; Sprengers, R.W.; Schutgens, R.E.; Slaper-Cortenbach, I.C.; van der Graaf, Y.; Algra, A.; van der Tweel, I.; Doevendans, P.A.; Mali, W.P.; Moll, F.L.; et al. Effect of repetitive intra-arterial infusion of bone marrow mononuclear cells in patients with no-option limb ischemia: The randomized, double-blind, placebo-controlled Rejuvenating Endothelial Progenitor Cells via Transcutaneous Intra-arterial Supplementation (JUVENTAS) trial. Circulation 2015, 131, 851–860. [Google Scholar] [PubMed]
  28. Fowkes, F.G.; Price, J.F. Gene therapy for critical limb ischaemia: The TAMARIS trial. Lancet 2011, 377, 1894–1896. [Google Scholar] [CrossRef]
  29. Forster, R.; Liew, A.; Bhattacharya, V.; Shaw, J.; Stansby, G. Gene therapy for peripheral arterial disease. Cochrane Database Syst. Rev. 2018, 10, CD012058. [Google Scholar] [CrossRef]
  30. Rigato, M.; Monami, M.; Fadini, G.P. Autologous Cell Therapy for Peripheral Arterial Disease: Systematic Review and Meta-Analysis of Randomized, Nonrandomized, and Noncontrolled Studies. Circ. Res. 2017, 120, 1326–1340. [Google Scholar] [CrossRef]
  31. Xie, B.; Luo, H.; Zhang, Y.; Wang, Q.; Zhou, C.; Xu, D. Autologous Stem Cell Therapy in Critical Limb Ischemia: A Meta-Analysis of Randomized Controlled Trials. Stem Cells Int. 2018, 2018, 7528464. [Google Scholar] [CrossRef] [PubMed]
  32. Gao, W.; Chen, D.; Liu, G.; Ran, X. Autologous stem cell therapy for peripheral arterial disease: A systematic review and meta-analysis of randomized controlled trials. Stem Cell Res. Ther. 2019, 10, 140. [Google Scholar] [CrossRef] [PubMed]
  33. Pu, H.; Huang, Q.; Zhang, X.; Wu, Z.; Qiu, P.; Jiang, Y.; Wang, R.; Zhao, Z.; Xu, Z.; Qin, J.; et al. A meta-analysis of randomized controlled trials on therapeutic efficacy and safety of autologous cell therapy for atherosclerosis obliterans. J. Vasc. Surg. 2022, 75, 1440–1449.e5. [Google Scholar] [CrossRef] [PubMed]
  34. Beltrán-Camacho, L.; Rojas-Torres, M.; Durán-Ruiz, M.C. Current Status of Angiogenic Cell Therapy and Related Strategies Applied in Critical Limb Ischemia. Int. J. Mol. Sci. 2021, 22, 2335. [Google Scholar] [CrossRef] [PubMed]
  35. Ruiz-Salmeron, R.; de la Cuesta-Diaz, A.; Constantino-Bermejo, M.; Pérez-Camacho, I.; Marcos-Sánchez, F.; Hmadcha, A.; Soria, B. Angiographic demonstration of neoangiogenesis after intra-arterial infusion of autologous bone marrow mononuclear cells in diabetic patients with critical limb ischemia. Cell Transpl. 2011, 20, 1629–1639. [Google Scholar] [CrossRef] [PubMed]
  36. De Angelis, B.; Gentile, P.; Orlandi, F.; Bocchini, I.; Di Pasquali, C.; Agovino, A.; Gizzi, C.; Patrizi, F.; Scioli, M.G.; Orlandi, A.; et al. Limb rescue: A new autologous-peripheral blood mononuclear cells technology in critical limb ischemia and chronic ulcers. Tissue Eng. Part C Methods 2015, 21, 423–435. [Google Scholar] [CrossRef]
  37. O’Neill, K.M.; Campbell, D.C.; Edgar, K.S.; Gill, E.K.; Moez, A.; McLoughlin, K.J.; O’Neill, C.L.; Dellett, M.; Hargey, C.J.; Abudalo, R.A.; et al. NOX4 is a major regulator of cord blood-derived endothelial colony-forming cells which promotes post-ischaemic revascularization. Cardiovasc. Res. 2020, 116, 393–405. [Google Scholar] [CrossRef]
  38. Hu, X.; Wu, R.; Jiang, Z.; Wang, L.; Chen, P.; Zhang, L.; Yang, L.; Wu, Y.; Chen, H.; Chen, H.; et al. Leptin signalling is required for augmented therapeutic properties of mesenchymal stem cells conferred by hypoxia preconditioning. Stem Cells 2014, 32, 2702–2713. [Google Scholar] [CrossRef]
  39. Ma, Q.; Xia, X.; Tao, Q.; Lu, K.; Shen, J.; Xu, Q.; Hu, X.; Tang, Y.; Block, N.L.; Webster, K.A.; et al. Profound Actions of an Agonist of Growth Hormone-Releasing Hormone on Angiogenic Therapy by Mesenchymal Stem Cells. Arter Thromb. Vasc. Biol. 2016, 36, 663–672. [Google Scholar] [CrossRef]
  40. Deev, R.V.; Bozo, I.Y.; Mzhavanadze, N.D.; Voronov, D.A.; Gavrilenko, A.V.; Chervyakov, Y.V.; Staroverov, I.N.; Kalinin, R.E.; Shvalb, P.G.; Isaev, A.A. pCMV-vegf165 Intramuscular Gene Transfer is an Effective Method of Treatment for Patients with Chronic Lower Limb Ischemia. J. Cardiovasc. Pharmacol. Ther. 2015, 20, 473–482. [Google Scholar] [CrossRef]
  41. Deev, R.; Plaksa, I.; Bozo, I.; Isaev, A. Results of an International Postmarketing Surveillance Study of pl-VEGF165 Safety and Efficacy in 210 Patients with Peripheral Arterial Disease. Am. J. Cardiovasc. Drugs 2017, 17, 235–242. [Google Scholar] [CrossRef] [PubMed]
  42. Deev, R.; Plaksa, I.; Bozo, I.; Mzhavanadze, N.; Suchkov, I.; Chervyakov, Y.; Staroverov, I.; Kalinin, R.; Isaev, A. Results of 5-year follow-up study in patients with peripheral artery disease treated with PL-VEGF165 for intermittent claudication. Ther. Adv. Cardiovasc. Dis. 2018, 12, 237–246. [Google Scholar] [CrossRef] [PubMed]
  43. Gu, Y.; Cui, S.; Wang, Q.; Liu, C.; Jin, B.; Guo, W.; Liu, C.; Chu, T.; Shu, C.; Zhang, F.; et al. A Randomized, Double-Blind, Placebo-Controlled Phase II Study of Hepatocyte Growth Factor in the Treatment of Critical Limb Ischemia. Mol. Ther. 2019, 27, 2158–2165. [Google Scholar] [CrossRef] [PubMed]
  44. Ylä-Herttuala, S. Gene Therapy of Critical Limb Ischemia Enters Clinical Use. Mol. Ther. 2019, 27, 2053. [Google Scholar] [CrossRef] [PubMed]
  45. Morishita, R.; Shimamura, M.; Takeya, Y.; Nakagami, H.; Chujo, M.; Ishihama, T.; Yamada, E.; Rakugi, H. Combined Analysis of Clinical Data on HGF Gene Therapy to Treat Critical Limb Ischemia in Japan. Curr. Gene Ther. 2020, 20, 25–35. [Google Scholar] [PubMed]
  46. Sanada, F.; Fujikawa, T.; Shibata, K.; Taniyama, Y.; Rakugi, H.; Morishita, R. Therapeutic Angiogenesis Using HGF Plasmid. Ann. Vasc. Dis. 2020, 13, 109–115. [Google Scholar] [CrossRef] [PubMed]
  47. Kessler, J.A.; Shaibani, A.; Sang, C.N.; Christiansen, M.; Kudrow, D.; Vinik, A.; Shin, N. Gene therapy for diabetic peripheral neuropathy: A randomized, placebo-controlled phase III study of VM202, a plasmid DNA encoding human hepatocyte growth factor. Clin. Transl. Sci. 2021, 14, 1176–1184. [Google Scholar] [CrossRef]
  48. Barć, P.; Antkiewicz, M.; Śliwa, B.; Frączkowska, K.; Guziński, M.; Dawiskiba, T.; Małodobra-Mazur, M.; Witkiewicz, W.; Kupczyńska, D.; Strzelec, B.; et al. Double VEGF/HGF Gene Therapy in Critical Limb Ischemia Complicated by Diabetes Mellitus. J. Cardiovasc. Transl. Res. 2021, 14, 409–415. [Google Scholar] [CrossRef]
  49. Shishehbor, M.H.; Rundback, J.; Bunte, M.; Hammad, T.A.; Miller, L.; Patel, P.D.; Sadanandan, S.; Fitzgerald, M.; Pastore, J.; Kashyap, V.; et al. SDF-1 plasmid treatment for patients with peripheral artery disease (STOP-PAD): Randomized, double-blind, placebo-controlled clinical trial. Vasc. Med. 2019, 24, 200–207. [Google Scholar] [CrossRef]
  50. Zaccagnini, G.; Gaetano, C.; Della Pietra, L.; Nanni, S.; Grasselli, A.; Mangoni, A.; Benvenuto, R.; Fabrizi, M.; Truffa, S.; Germani, A.; et al. Telomerase mediates vascular endothelial growth factor-dependent responsiveness in a rat model of hind limb ischemia. J. Biol. Chem. 2005, 280, 14790–14798. [Google Scholar] [CrossRef]
  51. Golledge, J. Update on the pathophysiology and medical treatment of peripheral artery disease. Nat. Rev. Cardiol. 2022, 19, 456–474. [Google Scholar] [CrossRef] [PubMed]
  52. Han, J.; Luo, L.; Marcelina, O.; Kasim, V.; Wu, S. Therapeutic angiogenesis-based strategy for peripheral artery disease. Theranostics 2022, 12, 5015–5033. [Google Scholar] [CrossRef] [PubMed]
  53. Rebar, E.J.; Huang, Y.; Hickey, R.; Nath, A.K.; Meoli, D.; Nath, S.; Chen, B.; Xu, L.; Liang, Y.; Jamieson, A.C.; et al. Induction of angiogenesis in a mouse model using engineered transcription factors. Nat. Med. 2002, 8, 1427–1432. [Google Scholar] [CrossRef] [PubMed]
  54. Campia, U.; Gerhard-Herman, M.; Piazza, G.; Goldhaber, S.Z. Peripheral Artery Disease: Past, Present, and Future. Am. J. Med. 2019, 132, 1133–1141. [Google Scholar] [CrossRef] [PubMed]
  55. Firnhaber, J.M.; Powell, C.S. Lower Extremity Peripheral Artery Disease: Diagnosis and Treatment. Am. Fam. Physician 2019, 99, 362–369. [Google Scholar] [PubMed]
  56. Boden, J.; Lassance-Soares, R.M.; Wang, H.; Wei, Y.; Spiga, M.G.; Adi, J.; Layman, H.; Yu, H.; Vazquez-Padron, R.I.; Andreopoulos, F.; et al. Vascular Regeneration in Ischemic Hindlimb by Adeno-Associated Virus Expressing Conditionally Silenced Vascular Endothelial Growth Factor. J. Am. Heart Assoc. 2016, 5, e001815. [Google Scholar] [CrossRef] [PubMed]
  57. Krishna, S.M.; Omer, S.M.; Golledge, J. Evaluation of the clinical relevance and limitations of current pre-clinical models of peripheral artery disease. Clin. Sci. 2016, 130, 127–150. [Google Scholar] [CrossRef] [PubMed]
  58. Krishna, S.M.; Omer, S.M.; Li, J.; Morton, S.K.; Jose, R.J.; Golledge, J. Development of a two-stage limb ischemia model to better simulate human peripheral artery disease. Sci. Rep. 2020, 10, 3449. [Google Scholar] [CrossRef]
  59. Golledge, J.; Fernando, M.E.; Armstrong, D.G. Current Management of Peripheral Artery Disease: Focus on Pharmacotherapy. Drugs 2022, 82, 1165–1177. [Google Scholar] [CrossRef]
  60. Annex, B.H. Therapeutic angiogenesis for critical limb ischaemia. Nat. Rev. Cardiol. 2013, 10, 387–396. [Google Scholar] [CrossRef]
  61. Peck, M.A.; Crawford, R.S.; Abularrage, C.J.; Patel, V.I.; Conrad, M.F.; Yoo, J.H.; Watkins, M.T.; Albadawi, H. A functional murine model of hindlimb demand ischemia. Ann. Vasc. Surg. 2010, 24, 532–537. [Google Scholar] [CrossRef] [PubMed]
  62. Lin, J.B.; Phillips, E.H.; Riggins, T.A.; Sangha, G.S.; Chakraborty, S.; Lee, J.Y.; Lycke, R.J.; Hernandez, C.L.; Soepriatna, A.H.; Thorne, B.R.; et al. Imaging of small animal peripheral artery disease models: Recent advancements and translational potential. Int. J. Mol. Sci. 2015, 16, 11131–11177. [Google Scholar] [CrossRef] [PubMed]
  63. Lotfi, S.; Patel, A.S.; Mattock, K.; Egginton, S.; Smith, A.; Modarai, B. Towards a more relevant hind limb model of muscle ischaemia. Atherosclerosis 2013, 227, 1–8. [Google Scholar] [CrossRef] [PubMed]
  64. Yan, K.; Zheng, J.; Zöllner, F.G.; Schwenke, K.; Pallavi, P.; Keese, M. A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision. J. Vis. Exp. 2021, 13, 171. [Google Scholar]
  65. Aref, Z.; de Vries, M.R.; Quax, P.H.A. Variations in Surgical Procedures for Inducing Hind Limb Ischemia in Mice and the Impact of These Variations on Neovascularization Assessment. Int. J. Mol. Sci. 2019, 20, 3704. [Google Scholar] [CrossRef] [PubMed]
  66. Shaked, Y.; Bertolini, F.; Man, S.; Rogers, M.S.; Cervi, D.; Foutz, T.; Rawn, K.; Voskas, D.; Dumont, D.J.; Ben-David, Y.; et al. Genetic heterogeneity of the vasculogenic phenotype parallels angiogenesis; Implications for cellular surrogate marker analysis of antiangiogenesis. Cancer Cell. 2005, 7, 101–111. [Google Scholar] [PubMed]
  67. Bosch-Marce, M.; Okuyama, H.; Wesley, J.B.; Sarkar, K.; Kimura, H.; Liu, Y.V.; Zhang, H.; Strazza, M.; Rey, S.; Savino, L.; et al. Effects of aging and hypoxia-inducible factor-1 activity on angiogenic cell mobilization and recovery of perfusion after limb ischemia. Circ. Res. 2007, 101, 1310–1318. [Google Scholar] [CrossRef] [PubMed]
  68. Westvik, T.S.; Fitzgerald, T.N.; Muto, A.; Maloney, S.P.; Pimiento, J.M.; Fancher, T.T.; Magri, D.; Westvik, H.H.; Nishibe, T.; Velazquez, O.C.; et al. Limb ischemia after iliac ligation in aged mice stimulates angiogenesis without arteriogenesis. J. Vasc. Surg. 2009, 49, 464–473. [Google Scholar] [CrossRef]
  69. Wang, J.; Peng, X.; Lassance-Soares, R.M.; Najafi, A.H.; Alderman, L.O.; Sood, S.; Xue, Z.; Chan, R.; Faber, J.E.; Epstein, S.E.; et al. Aging-induced collateral dysfunction: Impaired responsiveness of collaterals and susceptibility to apoptosis via dysfunctional eNOS signaling. J. Cardiovasc. Transl. Res. 2011, 4, 779–789. [Google Scholar] [CrossRef]
  70. Kuro-o, M.; Matsumura, Y.; Aizawa, H.; Kawaguchi, H.; Suga, T.; Utsugi, T.; Ohyama, Y.; Kurabayashi, M.; Kaname, T.; Kume, E.; et al. Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature 1997, 390, 45–51. [Google Scholar] [CrossRef]
  71. Fukino, K.; Suzuki, T.; Saito, Y.; Shindo, T.; Amaki, T.; Kurabayashi, M.; Nagai, R. Regulation of angiogenesis by the aging suppressor gene klotho. Biochem. Biophys. Res. Commun. 2002, 293, 332–337. [Google Scholar] [CrossRef] [PubMed]
  72. Abraham, C.R.; Li, A. Aging-suppressor Klotho: Prospects in diagnostics and therapeutics. Ageing Res Rev. 2022, 82, 101766. [Google Scholar] [CrossRef] [PubMed]
  73. Shimada, T.; Takeshita, Y.; Murohara, T.; Sasaki, K.; Egami, K.; Shintani, S.; Katsuda, Y.; Ikeda, H.; Nabeshima, Y.; Imaizumi, T. Angiogenesis and vasculogenesis are impaired in the precocious-aging klotho mouse. Circulation 2004, 110, 1148–1155. [Google Scholar] [CrossRef] [PubMed]
  74. Martín-Núñez, E.; Pérez-Castro, A.; Tagua, V.G.; Hernández-Carballo, C.; Ferri, C.; Pérez-Delgado, N.; Rodríguez-Ramos, S.; Cerro-López, P.; López-Castillo, Á.; Delgado-Molinos, A.; et al. Klotho expression in peripheral blood circulating cells is associated with vascular and systemic inflammation in atherosclerotic vascular disease. Sci Rep. 2022, 12, 8422. [Google Scholar] [CrossRef]
  75. Libby, P. The changing landscape of atherosclerosis. Nature 2021, 592, 524–533. [Google Scholar] [CrossRef]
  76. Poledniczek, M.; Neumayer, C.; Kopp, C.W.; Schlager, O.; Gremmel, T.; Jozkowicz, A.; Gschwandtner, M.E.; Koppensteiner, R.; Wadowski, P.P. Micro- and Macrovascular Effects of Inflammation in Peripheral Artery Disease-Pathophysiology and Translational Therapeutic Approaches. Biomedicines 2023, 11, 2284. [Google Scholar] [CrossRef]
  77. Krishna, S.M.; Moxon, J.V.; Golledge, J. A review of the pathophysiology and potential biomarkers for peripheral artery disease. Int. J. Mol. Sci. 2015, 16, 11294–11322. [Google Scholar] [CrossRef]
  78. McDermott, M.M.; Ferrucci, L.; Gonzalez-Freire, M.; Kosmac, K.; Leeuwenburgh, C.; Peterson, C.A.; Saini, S.; Sufit, R. Skeletal Muscle Pathology in Peripheral Artery Disease: A Brief Review. Arterioscler. Thromb. Vasc. Biol. 2020, 40, 2577–2585. [Google Scholar] [CrossRef]
  79. Burgmaier, M.; Schutters, K.; Willems, B.; van der Vorst, E.P.; Kusters, D.; Chatrou, M.; Norling, L.; Biessen, E.A.; Cleutjens, J.; Perretti, M.; et al. AnxA5 reduces plaque inflammation of advanced atherosclerotic lesions in apoE(-/-) mice. J. Cell Mol. Med. 2014, 18, 2117–2124. [Google Scholar] [CrossRef]
  80. Xiong, W.; Wang, X.; Dai, D.; Zhang, B.; Lu, L.; Tao, R. The anti-inflammatory vasostatin-2 attenuates atherosclerosis in ApoE-/- mice and inhibits monocyte/macrophage recruitment. Thromb. Haemost. 2017, 117, 401–414. [Google Scholar] [CrossRef]
  81. Kahles, F.; Liberman, A.; Halim, C.; Rau, M.; Möllmann, J.; Mertens, R.W.; Rückbeil, M.; Diepolder, I.; Walla, B.; Diebold, S.; et al. The incretin hormone GIP is upregulated in patients with atherosclerosis and stabilizes plaques in ApoE-/- mice by blocking monocyte/macrophage activation. Mol. Metab. 2018, 14, 150–157. [Google Scholar] [CrossRef] [PubMed]
  82. Xu, Q.; Wang, J.; He, J.; Zhou, M.; Adi, J.; Webster, K.A.; Yu, H. Impaired CXCR4 expression and cell engraftment of bone marrow-derived cells from aged atherogenic mice. Atherosclerosis 2011, 219, 92–99. [Google Scholar] [CrossRef] [PubMed]
  83. Qin, H.; Liu, P.; Lin, S. Effects of Astragaloside IV on the SDF-1/CXCR4 Expression in Atherosclerosis of apoE(-/-) Mice Induced by Hyperlipaemia. Evid. Based Complement. Altern. Med. 2015, 2015, 385154. [Google Scholar] [CrossRef]
  84. Heidt, T.; Sager, H.B.; Courties, G.; Dutta, P.; Iwamoto, Y.; Zaltsman, A.; von Zur Muhlen, C.; Bode, C.; Fricchione, G.L.; Denninger, J.; et al. Chronic variable stress activates hematopoietic stem cells. Nat. Med. 2014, 20, 754–758. [Google Scholar] [CrossRef] [PubMed]
  85. Schmitt, M.M.; Megens, R.T.; Zernecke, A.; Bidzhekov, K.; van den Akker, N.M.; Rademakers, T.; van Zandvoort, M.A.; Hackeng, T.M.; Koenen, R.R.; Weber, C. Endothelial junctional adhesion molecule-a guides monocytes into flow-dependent predilection sites of atherosclerosis. Circulation 2014, 129, 66–76. [Google Scholar] [CrossRef] [PubMed]
  86. Seo, H.S.; Lombardi, D.M.; Polinsky, P.; Powell-Braxton, L.; Bunting, S.; Schwartz, S.M.; Rosenfeld, M.E. Peripheral vascular stenosis in apolipoprotein E-deficient mice. Potential roles of lipid deposition, medial atrophy, and adventitial inflammation. Arterioscler. Thromb. Vasc. Biol. 1997, 17, 3593–3601. [Google Scholar] [CrossRef]
  87. Rodriguez-Menocal, L.; Wei, Y.; Pham, S.M.; St-Pierre, M.; Li, S.; Webster, K.A.; Goldschmidt-Clermont, P.; Vazquez-Padron, R.I. A novel mouse model of in-stent restenosis. Atherosclerosis 2010, 209, 359–366. [Google Scholar] [CrossRef]
  88. Cesar, L.; Suarez, S.V.; Adi, J.; Adi, N.; Vazquez-Padron, R.; Yu, H.; Ma, Q.; Goldschmidt-Clermont, P.J.; Agatston, A.; Kurlansky, P.; et al. An essential role for diet in exercise-mediated protection against dyslipidemia, inflammation, and atherosclerosis in ApoE-/-mice. PLoS ONE 2011, 6, e17263. [Google Scholar] [CrossRef]
  89. Balestrieri, M.L.; Lu, S.J.; de Nigris, F.; Giovane, A.; Williams-Ignarro, S.; D’Armiento, F.P.; Napoli, C. Therapeutic angiogenesis in diabetic apolipoprotein E-deficient mice using bone marrow cells, functional hemangioblasts and metabolic intervention. Atherosclerosis 2010, 209, 403–414. [Google Scholar] [CrossRef]
  90. Kang, J.; Albadawi, H.; Patel, V.I.; Abbruzzese, T.A.; Yoo, J.H.; Austen WGJr Watkins, M.T. Apolipoprotein E-/- mice have delayed skeletal muscle healing after hind limb ischemia-reperfusion. J. Vasc. Surg. 2008, 48, 701–708. [Google Scholar] [CrossRef] [PubMed]
  91. Couffinhal, T.; Silver, M.; Kearney, M.; Sullivan, A.; Witzenbichler, B.; Magner, M.; Annex, B.; Peters, K.; Isner, J.M. Impaired collateral vessel development associated with reduced expression of vascular endothelial growth factor in ApoE-/- mice. Circulation 1999, 99, 3188–3198. [Google Scholar] [CrossRef] [PubMed]
  92. Xie, D.; Li, Y.; Reed, E.A.; Odronic, S.I.; Kontos, C.D.; Annex, B.H. An engineered vascular endothelial growth factor-activating transcription factor induces therapeutic angiogenesis in ApoE knockout mice with hindlimb ischemia. J. Vasc. Surg. 2006, 44, 166–175. [Google Scholar] [CrossRef] [PubMed]
  93. Lejay, A.; Charles, A.L.; Georg, I.; Goupilleau, F.; Delay, C.; Talha, S.; Thaveau, F.; Chakfé, N.; Geny, B. Critical Limb Ischaemia Exacerbates Mitochondrial Dysfunction in ApoE-/- Mice Compared with ApoE+/+ Mice, but N-acetyl Cysteine still Confers Protection. Eur. J. Vasc. Endovasc. Surg. 2019, 58, 576–582. [Google Scholar] [CrossRef] [PubMed]
  94. Feinberg, M.W.; Moore, K.J. MicroRNA Regulation of Atherosclerosis. Circ. Res. 2016, 118, 703–720. [Google Scholar] [CrossRef] [PubMed]
  95. Shan, Z.; Yao, C.; Li, Z.L.; Teng, Y.; Li, W.; Wang, J.S.; Ye, C.S.; Chang, G.Q.; Huang, X.L.; Li, X.X.; et al. Differentially expressed microRNAs at different stages of atherosclerosis in ApoE-deficient mice. Chin. Med. J. 2013, 126, 515–520. [Google Scholar] [CrossRef]
  96. Vogiatzi, G.; Oikonomou, E.; Deftereos, S.; Siasos, G.; Tousoulis, D. Peripheral artery disease: A micro-RNA-related condition? Curr. Opin. Pharmacol. 2018, 39, 105–112. [Google Scholar] [CrossRef]
  97. Pérez-Cremades, D.; Cheng, H.S.; Feinberg, M.W. Noncoding RNAs in Critical Limb Ischemia. Arterioscler. Thromb. Vasc. Biol. 2020, 40, 523–533. [Google Scholar] [CrossRef]
  98. Gao, L.; Zeng, H.; Zhang, T.; Mao, C.; Wang, Y.; Han, Z.; Chen, K.; Zhang, J.; Fan, Y.; Gu, J.; et al. MicroRNA-21 deficiency attenuated atherogenesis and decreased macrophage infiltration by targeting Dusp-8. Atherosclerosis 2019, 291, 78–86. [Google Scholar] [CrossRef]
  99. Rotllan, N.; Price, N.; Pati, P.; Goedeke, L.; Fernández-Hernando, C. microRNAs in lipoprotein metabolism and cardiometabolic disorders. Atherosclerosis 2016, 246, 352–360. [Google Scholar] [CrossRef]
  100. Desjarlais, M.; Dussault, S.; Rivard, F.; Harel, S.; Sanchez, V.; Hussain, S.N.A.; Rivard, A. Forced expression of microRNA-146b reduces TRAF6-dependent inflammation and improves ischemia-induced neovascularization in hypercholesterolemic conditions. Atherosclerosis 2019, 289, 73–84. [Google Scholar] [CrossRef] [PubMed]
  101. Qun, L.; Wenda, X.; Weihong, S.; Jianyang, M.; Wei, C.; Fangzhou, L.; Zhenyao, X.; Pingjin, G. miRNA-27b modulates endothelial cell angiogenesis by directly targeting Naa15 in atherogenesis. Atherosclerosis 2016, 254, 184–192. [Google Scholar] [CrossRef] [PubMed]
  102. Wang, J.; Peng, H.; Timur, A.A.; Pasupuleti, V.; Yao, Y.; Zhang, T.; You, S.A.; Fan, C.; Yu, Y.; Jia, X.; et al. Receptor and Molecular Mechanism of AGGF1 Signaling in Endothelial Cell Functions and Angiogenesis. Arterioscler. Thromb. Vasc. Biol. 2021, 41, 2756–2769. [Google Scholar] [CrossRef] [PubMed]
  103. Li, L.; Chen, D.; Li, J.; Wang, X.; Wang, N.; Xu, C.; Wang, Q.K. Aggf1 acts at the top of the genetic regulatory hierarchy in specification of hemangioblasts in zebrafish. Blood 2014, 123, 501–508. [Google Scholar] [CrossRef] [PubMed]
  104. Fan, C.; Chen, Q.; Wang, Q.K. Functional role of transcriptional factor TBX5 in pre-mRNA splicing and Holt-Oram syndrome via association with SC35. J. Biol. Chem. 2009, 284, 25653–25663. [Google Scholar] [CrossRef]
  105. Zhang, T.; Yao, Y.; Wang, J.; Li, Y.; He, P.; Pasupuleti, V.; Hu, Z.; Jia, X.; Song, Q.; Tian, X.L.; et al. Haploinsufficiency of Klippel-Trenaunay syndrome gene Aggf1 inhibits developmental and pathological angiogenesis by inactivating PI3K and AKT and disrupts vascular integrity by activating VE-cadherin. Hum. Mol. Genet. 2016, 25, 5094–5110. [Google Scholar] [PubMed]
  106. Fan, C.; Ouyang, P.; Timur, A.A.; He, P.; You, S.A.; Hu, Y.; Ke, T.; Driscoll, D.J.; Chen, Q.; Wang, Q.K. Novel roles of GATA1 in regulation of angiogenic factor AGGF1 and endothelial cell function. J. Biol. Chem. 2009, 284, 23331–23343. [Google Scholar] [CrossRef]
  107. Tian, X.L.; Kadaba, R.; You, S.A.; Liu, M.; Timur, A.A.; Yang, L.; Chen, Q.; Szafranski, P.; Rao, S.; Wu, L.; et al. Identification of an angiogenic factor that when mutated causes susceptibility to Klippel-Trenaunay syndrome. Nature 2004, 427, 640–645. [Google Scholar] [CrossRef] [PubMed]
  108. Lu, Q.; Yao, Y.; Yao, Y.; Liu, S.; Huang, Y.; Lu, S.; Bai, Y.; Zhou, B.; Xu, Y.; Li, L.; et al. Angiogenic factor AGGF1 promotes therapeutic angiogenesis in a mouse limb ischemia model. PLoS ONE 2012, 7, e46998. [Google Scholar] [CrossRef] [PubMed]
  109. Mac Gabhann, F.; Annex, B.H. AGGF1 Shows the α5β1 Integrin to Be Another Akt-or in a Common Angiogenesis Scene. Arterioscler Thromb. Vasc. Biol. 2021, 41, 2770–2772. [Google Scholar] [CrossRef] [PubMed]
  110. Yao, Y.; Li, Y.; Song, Q.; Hu, C.; Xie, W.; Xu, C.; Chen, Q.; Wang, Q.K. Angiogenic Factor AGGF1-Primed Endothelial Progenitor Cells Repair Vascular Defect in Diabetic Mice. Diabetes 2019, 68, 1635–1648. [Google Scholar] [CrossRef]
  111. Yu, Y.; Li, Y.; Peng, H.; Song, Q.; Da, X.; Li, H.; He, Z.; Ren, X.; Xu, C.; Yao, Y.; et al. Angiogenic factor AGGF1 blocks neointimal formation after vascular injury via interaction with integrin α7 on vascular smooth muscle cells. J. Biol. Chem. 2022, 298, 101759. [Google Scholar] [CrossRef] [PubMed]
  112. Harper, S.J.; Bates, D.O. VEGF-A splicing: The key to anti-angiogenic therapeutics? Nat. Rev. Cancer 2008, 8, 880–887. [Google Scholar] [CrossRef] [PubMed]
  113. Rennel, E.S.; Varey, A.H.; Churchill, A.J.; Wheatley, E.R.; Stewart, L.; Mather, S.; Bates, D.O.; Harper, S.J. VEGF(121)b, a new member of the VEGF(xxx)b family of VEGF-A splice isoforms, inhibits neovascularisation and tumour growth in vivo. Br. J. Cancer 2009, 101, 1183–1193. [Google Scholar] [CrossRef] [PubMed]
  114. Catena, R.; Larzabal, L.; Larrayoz, M.; Molina, E.; Hermida, J.; Agorreta, J.; Montes, R.; Pio, R.; Montuenga, L.M.; Calvo, A. VEGF121b and VEGF165b are weakly angiogenic isoforms of VEGF-A. Mol. Cancer 2010, 9, 320. [Google Scholar] [CrossRef] [PubMed]
  115. Kawamura, H.; Li, X.; Harper, S.J.; Bates, D.O.; Claesson-Welsh, L. Vascular endothelial growth factor (VEGF)-A165b is a weak in vitro agonist for VEGF receptor-2 due to lack of coreceptor binding and deficient regulation of kinase activity. Cancer Res. 2008, 68, 4683–4692. [Google Scholar] [CrossRef] [PubMed]
  116. Chamorro-Jorganes, A.; Araldi, E.; Penalva, L.O.; Sandhu, D.; Fernández-Hernando, C.; Suárez, Y. MicroRNA-16 and microRNA-424 regulate cell-autonomous angiogenic functions in endothelial cells via targeting vascular endothelial growth factor receptor-2 and fibroblast growth factor receptor-1. Arterioscler. Thromb. Vasc. Biol. 2011, 31, 2595–2606. [Google Scholar] [CrossRef]
  117. Yue, J.; Tigyi, G. Conservation of miR-15a/16-1 and miR-15b/16-2 clusters. Mamm. Genome 2010, 21, 88–94. [Google Scholar] [CrossRef]
  118. Spinetti, G.; Fortunato, O.; Caporali, A.; Shantikumar, S.; Marchetti, M.; Meloni, M.; Descamps, B.; Floris, I.; Sangalli, E.; Vono, R.; et al. MicroRNA-15a and microRNA-16 impair human circulating proangiogenic cell functions and are increased in the proangiogenic cells and serum of patients with critical limb ischemia. Circ. Res. 2013, 112, 335–346. [Google Scholar] [CrossRef]
  119. Yin, K.J.; Olsen, K.; Hamblin, M.; Zhang, J.; Schwendeman, S.P.; Chen, Y.E. Vascular endothelial cell-specific microRNA-15a inhibits angiogenesis in hindlimb ischemia. J. Biol. Chem. 2012, 287, 27055–27064. [Google Scholar] [CrossRef]
  120. Desjarlais, M.; Dussault, S.; Dhahri, W.; Mathieu, R.; Rivard, A. MicroRNA-150 Modulates Ischemia-Induced Neovascularization in Atherosclerotic Conditions. Arterioscler. Thromb. Vasc. Biol. 2017, 37, 900–908. [Google Scholar] [CrossRef]
  121. Ryan, T.E.; Schmidt, C.A.; Tarpey, M.D.; Amorese, A.J.; Yamaguchi, D.J.; Goldberg, E.J.; Iñigo, M.M.; Karnekar, R.; O’Rourke, A.; Ervasti, J.M.; et al. PFKFB3-mediated glycolysis rescues myopathic outcomes in the ischemic limb. JCI Insight 2020, 5, e139628. [Google Scholar] [CrossRef]
  122. Gomes de Almeida Schirmer, B.; Crucet, M.; Stivala, S.; Vucicevic, G.; da Silva Barcelos, L.; Vanhoutte, P.M.; Pellegrini, G.; Camici, G.G.; Seebeck, P.; Pfundstein, S.; et al. The NO-donor MPC-1011 stimulates angiogenesis and arteriogenesis and improves hindlimb ischemia via a cGMP-dependent pathway involving VEGF and SDF-1α. Atherosclerosis 2020, 304, 30–38. [Google Scholar] [CrossRef] [PubMed]
  123. Loffredo, L.; Pignatelli, P.; Cangemi, R.; Andreozzi, P.; Panico, M.A.; Meloni, V.; Violi, F. Imbalance between nitric oxide generation and oxidative stress in patients with peripheral arterial disease: Effect of an antioxidant treatment. J. Vasc. Surg. 2006, 44, 525–530. [Google Scholar] [CrossRef] [PubMed]
  124. Allen, J.D.; Giordano, T.; Kevil, C.G. Nitrite and nitric oxide metabolism in peripheral artery disease. Nitric Oxide 2012, 26, 217–222. [Google Scholar] [CrossRef] [PubMed]
  125. Hong, F.F.; Liang, X.Y.; Liu, W.; Lv, S.; He, S.J.; Kuang, H.B.; Yang, S.L. Roles of eNOS in atherosclerosis treatment. Inflamm. Res. 2019, 68, 429–441. [Google Scholar] [CrossRef] [PubMed]
  126. Ismaeel, A.; Papoutsi, E.; Miserlis, D.; Lavado, R.; Haynatzki, G.; Casale, G.P.; Bohannon, W.T.; Smith, R.S.; Eidson, J.L.; Brumberg, R.; et al. The Nitric Oxide System in Peripheral Artery Disease: Connection with Oxidative Stress and Biopterins. Antioxidants 2020, 9, 590. [Google Scholar] [CrossRef] [PubMed]
  127. Woessner, M.; VanBruggen, M.D.; Pieper, C.F.; Sloane, R.; Kraus, W.E.; Gow, A.J.; Allen, J.D. Beet the Best? Circ. Res. 2018, 123, 654–659. [Google Scholar] [CrossRef]
  128. Kenjale, A.A.; Ham, K.L.; Stabler, T.; Robbins, J.L.; Johnson, J.L.; Vanbruggen, M.; Privette, G.; Yim, E.; Kraus, W.E.; Allen, J.D. Dietary nitrate supplementation enhances exercise performance in peripheral arterial disease. J. Appl. Physiol. 2011, 110, 1582–1591. [Google Scholar] [CrossRef]
  129. Loffredo, L.; Perri, L.; Catasca, E.; Pignatelli, P.; Brancorsini, M.; Nocella, C.; De Falco, E.; Bartimoccia, S.; Frati, G.; Carnevale, R.; et al. Dark chocolate acutely improves walking autonomy in patients with peripheral artery disease. J. Am. Heart Assoc. 2014, 3, e001072. [Google Scholar] [CrossRef]
  130. Loffredo, L.; Perri, L.; Catasca, E.; Pignatelli, P.; Brancorsini, M.; Nocella, C.; De Falco, E.; Bartimoccia, S.; Frati, G.; Carnevale, R.; et al. Cocoa to Improve Walking Performance in Older People with Peripheral Artery Disease: The COCOA-PAD Pilot Randomized Clinical Trial. Circ. Res. 2020, 126, 589–599. [Google Scholar]
  131. Park, S.Y.; Pekas, E.J.; Headid, R.J., 3rd; Son, W.M.; Wooden, T.K.; Song, J.; Layec, G.; Yadav, S.K.; Mishra, P.K.; Pipinos, I.I. Acute mitochondrial antioxidant intake improves endothelial function, antioxidant enzyme activity, and exercise tolerance in patients with peripheral artery disease. Am. J. Physiol. Heart Circ. Physiol. 2020, 319, H456–H467. [Google Scholar] [CrossRef] [PubMed]
  132. Kotalczyk, A.; Vallabhaneni, S.R.; Lip, G.Y.H. Review new concepts in pharmacotherapy for peripheral arterial disease. Curr. Opin. Cardiol. 2021, 36, 720–726. [Google Scholar] [CrossRef] [PubMed]
  133. Omarjee, L.; Le Pabic, E.; Custaud, M.A.; Fontaine, C.; Locher, C.; Renault, A.; Jaquinandi, V.; Azzola, V.; Barbeau-Terrier, C.; Laporte, I.; et al. Effects of sildenafil on maximum walking time in patients with arterial claudication: The ARTERIOFIL study. Vasc. Pharmacol 2019, 118–119, 106563. [Google Scholar] [CrossRef] [PubMed]
  134. Kokkinidis, D.G.; Arfaras-Melainis, A.; Giannopoulos, S.; Katsaros, I.; Jawaid, O.; Jonnalagadda, A.K.; Parikh, S.A.; Secemsky, E.A.; Giri, J.; Kumbhani, D.J.; et al. Statin therapy for reduction of cardiovascular and limb-related events in critical limb ischemia: A systematic review and meta-analysis. Vasc Med. 2020, 25, 106–117. [Google Scholar] [CrossRef] [PubMed]
  135. Braun, S.K.; Jorge, D.W.; Bortolanza, G.; da Rocha, J.B.T. Effects of statin use on primary patency, mortality, and limb loss in patients undergoing lower-limb arterial angioplasty: A systematic review and meta-analysis. Int. J. Clin. Pharm. 2023, 45, 17–25. [Google Scholar] [CrossRef]
  136. Nissen, S.E.; Tuzcu, E.M.; Brewer, H.B.; Sipahi, I.; Nicholls, S.J.; Ganz, P.; Schoenhagen, P.; Waters, D.D.; Pepine, C.J.; Crowe, T.D.; et al. Effect of ACAT inhibition on the progression of coronary atherosclerosis. N. Engl. J. Med. 2006, 354, 1253–1263. [Google Scholar] [CrossRef]
  137. Hai, Q.; Smith, J.D. Acyl-Coenzyme A: Cholesterol Acyltransferase (ACAT) in Cholesterol Metabolism: From Its Discovery to Clinical Trials and the Genomics Era. Metabolites 2021, 11, 543. [Google Scholar] [CrossRef]
  138. Zhang, X.; Wang, Z.; Zheng, Y.; Yu, Q.; Zeng, M.; Bai, L.; Yang, L.; Guo, M.; Jiang, X.; Gan, J. Inhibitors of the NLRP3 inflammasome pathway as promising therapeutic candidates for inflammatory diseases (Review). Int. J. Mol. Med. 2023, 51, 35. [Google Scholar] [CrossRef]
  139. Borrego, A.; Colombo, F.; de Souza, J.G.; Jensen, J.R.; Dassano, A.; Piazza, R.; Rodrigues Dos Santos, B.A.; Ribeiro, O.G.; De Franco, M.; Cabrera, W.H.K.; et al. Pycard and BC017158 Candidate Genes of Irm1 Locus Modulate Inflammasome Activation for IL-1β Production. Front. Immunol. 2022, 13, 899569. [Google Scholar] [CrossRef]
  140. Ritchey, B.; Hai, Q.; Han, J.; Barnard, J.; Smith, J.D. Genetic variant in 3′ untranslated region of the mouse pycard gene regulates inflammasome activity. eLife 2021, 10, e68203. [Google Scholar] [CrossRef]
  141. Dougherty, C.J.; Smith, G.W.; Dorey, C.K.; Prentice, H.M.; Webster, K.A.; Blanks, J.C. Robust hypoxia-selective regulation of a retinal pigment epithelium-specific adeno-associated virus vector. Mol. Vis. 2008, 14, 471–480. [Google Scholar] [PubMed]
  142. Webster, K.A. Therapeutic angiogenesis for coronary artery disease: Clinical trials of proteins, plasmids, adenovirus, and stem cells. Future Cardiol. 2005, 1, 99–109. [Google Scholar] [CrossRef] [PubMed]
  143. Silver, E.; Argiro, A.; Hong, K.; Adler, E. Gene therapy vector-related myocarditis. Int. J. Cardiol. 2023, 131617. [Google Scholar] [CrossRef] [PubMed]
  144. Naso, M.F.; Tomkowicz, B.; Perry, W.L., 3rd; Strohl, W.R. Adeno-Associated Virus (AAV) as a Vector for Gene Therapy. BioDrugs 2017, 31, 317–334. [Google Scholar] [CrossRef]
  145. Mendell, J.R.; Al-Zaidy, S.A.; Rodino-Klapac, L.R.; Goodspeed, K.; Gray, S.J.; Kay, C.N.; Boye, S.L.; Boye, S.E.; George, L.A.; Salabarria, S.; et al. Current Clinical Applications of In Vivo Gene Therapy with AAVs. Mol. Ther. 2021, 29, 464–488. [Google Scholar] [CrossRef]
Genes 15 00135 g001
Figure 1. Gene and cell targets of therapeutic angiogenesis. Green boxes show target pro-angiogenic genes that have been tested in Phase I-III RCTs delivered IA or IM as proteins, cDNA plasmids or cDNA adenovirus. Yellow boxes indicate autologous cells also delivered IA or IM. Aqua boxes indicate ongoing trials of combination genes including two isoforms of human HGF (pHGF728 and pHGF723) and a bicistronic plasmid vector expressing human VEGF165 and HGF (pIRES/VEGF165/HGF). Unboxed denotes ongoing RCTs with viral delivery of human FGF2 by IM of Sendai virus and IM injections of human telomerase reverse transcriptase (hTERT) by AAV.
Figure 1. Gene and cell targets of therapeutic angiogenesis. Green boxes show target pro-angiogenic genes that have been tested in Phase I-III RCTs delivered IA or IM as proteins, cDNA plasmids or cDNA adenovirus. Yellow boxes indicate autologous cells also delivered IA or IM. Aqua boxes indicate ongoing trials of combination genes including two isoforms of human HGF (pHGF728 and pHGF723) and a bicistronic plasmid vector expressing human VEGF165 and HGF (pIRES/VEGF165/HGF). Unboxed denotes ongoing RCTs with viral delivery of human FGF2 by IM of Sendai virus and IM injections of human telomerase reverse transcriptase (hTERT) by AAV.
Genes 15 00135 g001
Table 1. RCTs from 2000–2007 used for meta-analysis. For a complete list of studies see De Haro et al. [12] and Iyer and Annex [7]. IA: intra-arterial; IM: intramuscular; N/C: no significant change; QoL: quality of life. rFGF2: recombinant fibroblast growth factor 2. All treatments are deemed safe with indication of efficacy in rFGF-2 and BMMNC trials.
Table 1. RCTs from 2000–2007 used for meta-analysis. For a complete list of studies see De Haro et al. [12] and Iyer and Annex [7]. IA: intra-arterial; IM: intramuscular; N/C: no significant change; QoL: quality of life. rFGF2: recombinant fibroblast growth factor 2. All treatments are deemed safe with indication of efficacy in rFGF-2 and BMMNC trials.
AuthorPhaseTreatmentTreatment/CTRLMajor Findings
Lazarous et al. [13]IIA rFGF-213/6Safe. Increased calf blood flow at 6 months in treatments.
Rajagopalan et al. [14]
(RAVE)		IIIM Ad2-
VEGF121		15/3Safe. N/C ABI, PFWT, QoL.
Lederman et al. [15]
(TRAFFIC)		IIIA rFGF-2116/58Safe. Improved PFWT at 90 days; early improved ABI.
Makinen et al. [16]IIIA Ad2- and pVEGF16535/19Ad antibodies. Improved vascularity both treatments; N/C ABI or Rutherford class vs control.
Grossman et al. [17]
(DELTA)		IIIM pDel-1 +
poloxamer 188		52/53N/C PFWT ABI, claudication compared with control poloxamer 188 alone.
Tateishi-Yuyama et al. [18] (TACT)IIIM BMMNC or PBMNC25 unilateral
22 bilateral		Safe. Improved ABI, TcO2, PWT, increased collateral vessels in BM-MNC vs. PBMNC.
Table 2. RCTs from 2008–2015 used for meta-analysis. For a complete list of studies see Hammer A, Steiner S [19] and Iyer and Annex [7]. IA: intra-arterial; IM: intramuscular; N/C: no significant change; QoL: quality of life. rFGF2: recombinant fibroblast growth factor 2. All treatments are deemed safe with indications of efficacy especially in pHGF and BMMNC trials.
Table 2. RCTs from 2008–2015 used for meta-analysis. For a complete list of studies see Hammer A, Steiner S [19] and Iyer and Annex [7]. IA: intra-arterial; IM: intramuscular; N/C: no significant change; QoL: quality of life. rFGF2: recombinant fibroblast growth factor 2. All treatments are deemed safe with indications of efficacy especially in pHGF and BMMNC trials.
AuthorPhaseTreatmentTreatment/CNTRMajor Findings
Belch et al. [20] (TAMARIS)IIIIM pFGF1259/256N/C amputation or death
Van Huyan et al. [21]IIIA + IM
BMMNC		12/15Improved PFWT and ABI
Powell et al. [22]
(HGF STAT)		IIIM pHGF56/23Increased TcPO2; N/C TBI, ABI, wound healing
Nikol et al. [23]
(TALISMAN)		IIIM pFGF159/66Improved rest pain, QoL, amputation; N/C wound healing
Shigematsu et al. [24]IIIIM pHGF27/13Improved rest pain, ulcer size, QoL; N/C ABI or amputation
Creager et al. [25]IIIM AdHIF1α213/76N/C PFWT, QoL, ABI
Walter et al. [26]
(PROVASA)		IIIA BMMNC19/21Improved ulcer healing, rest pain, N/C ABI, amputation, death
Teraa et al. [27]
(JUVENTAS) (2015)		IIIA EPC81/79N/C amputation, death, ABI, ulcer size, QoL, rest pain, TcPO2
Table 3. Meta-analyses of autologous cell therapy conclude that the procedures are safe with evidence of efficacy on multiple outcomes including reduced amputation rates and QoL parameters. Variability of methods, reporting, and quality of data do not yet allow a determination on suitability for translation to clinical practice.
Table 3. Meta-analyses of autologous cell therapy conclude that the procedures are safe with evidence of efficacy on multiple outcomes including reduced amputation rates and QoL parameters. Variability of methods, reporting, and quality of data do not yet allow a determination on suitability for translation to clinical practice.
AuthorTitleMajor Findings
Rigato et al. [30] (2017)Autologous Cell Therapy for Peripheral Arterial Disease: Systematic Review and Meta-Analysis of Randomized, Nonrandomized, and Noncontrolled Studies.Autologous cell therapy may reduce the risk of major amputation, improve the probability of wound healing, and amputation-free survival, ameliorate pain and functional capacity. Results of the primary analysis were confirmed and strengthened by secondary analysis. No change in all-cause mortality.
Xie et al. [31] (2018) Autologous Stem Cell Therapy in Critical Limb Ischemia: A Meta-Analysis of Randomized Controlled Trials.Cell therapy significantly increased the probability of ulcer healing, angiogenesis, and reduced amputation rates. ABI and PFWT were significantly improved. Higher quality and larger RCTs are required to support clinical application.
Gao et al. [32] (2019) Autologous stem cell therapy for peripheral arterial disease: a systematic review and meta-analysis of randomized controlled trials.Improved healing rate of ulcers, ABI, TcO2, and PFWT; reduction in amputation rate and rest pain scores, no significant improvement in major limb salvage. High risk of bias and low-quality evidence of outcomes. Larger, placebo controlled, RCT are needed.
Pu et al. [33] (2022) A meta-analysis of randomized controlled trials on therapeutic efficacy and safety of autologous cell therapy for atherosclerosis obliterans.No-option CLI patients show significantly improved total amputation, major amputation, ABI, TcO2, and rest pain score compared with standard care. No effect on all-cause death or ulcer size.
Beltrán-Camacho et al. [34] (2021)Current Status of Angiogenic Cell Therapy and Related Strategies Applied in Critical Limb IschemiaCell therapy may represent an alternative for no-option CLI. Variability between trials is high, reflecting a lack of consensus on cell dose, cell types or sources, administration routes, parameters to define outcome efficacy, or cohorts themselves. Further investigation is required to better understand mechanism. Much work is needed to translate to clinical practice.
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Webster, K.A. Translational Relevance of Advanced Age and Atherosclerosis in Preclinical Trials of Biotherapies for Peripheral Artery Disease. Genes 2024, 15, 135. https://doi.org/10.3390/genes15010135

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Webster KA. Translational Relevance of Advanced Age and Atherosclerosis in Preclinical Trials of Biotherapies for Peripheral Artery Disease. Genes. 2024; 15(1):135. https://doi.org/10.3390/genes15010135

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Webster, Keith A. 2024. "Translational Relevance of Advanced Age and Atherosclerosis in Preclinical Trials of Biotherapies for Peripheral Artery Disease" Genes 15, no. 1: 135. https://doi.org/10.3390/genes15010135

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