Circulation Research is available at www.ahajournals.org/journal/res
Correspondence to: Nicholas J. Leeper, MD, Division of Vascular Surgery, Stanford University, 300 Pasteur Dr, Room H3638, Stanford, CA 94305. Email nleeper@ stanford.edu
For Sources of Funding and Disclosures, see page 1939. © 2021 American Heart Association, Inc.
PERIPHERAL VASCULAR DISEASE COMPENDIUM
Bench-to-Bedside in Vascular Medicine
Optimizing the Translational Pipeline for Patients With Peripheral Artery Disease
Tom Alsaigh, Belinda A. Di Bartolo, Jocelyne Mulangala, Gemma A. Figtree , Nicholas J. Leeper
ABSTRACT: Peripheral arterial disease is a growing worldwide problem with a wide spectrum of clinical severity and is projected to consume >$21 billion per year in the United States alone. While vascular researchers have brought several therapies to the clinic in recent years, few of these approaches have leveraged advances in high-throughput discovery screens, novel translational models, or innovative trial designs. In the following review, we discuss recent advances in unbiased genomics and broader omics technology platforms, along with preclinical vascular models designed to enhance our understanding of disease pathobiology and prioritize targets for additional investigation. Furthermore, we summarize novel approaches to clinical studies in subjects with claudication and ischemic ulceration, with an emphasis on streamlining and accelerating bench-to-bedside translation. By providing a framework designed to enhance each aspect of future clinical development programs, we hope to enrich the pipeline of therapies that may prevent loss of life and limb for those with peripheral arterial disease.
Key Words: atherosclerosis ◼ cardiology ◼ endothelial cell ◼ genomics ◼ peripheral arterial disease
O
ver 200 million people worldwide haveperiph-eral arterial disease (PAD), commonly defined as atherosclerosis involving arteries outside of the coronary circulation.1–3 The high incidence of lower
extremity PAD places an enormous burden on health care systems worldwide, necessitating novel strategies to curb disease progression.4 Affected patients suffer
from significant morbidities, including diminished blood supply to the limbs leading to claudication, critical limb ischemia, ulcer development, gangrene, and ultimately limb loss. While PAD treatments aimed at prevent-ing progression of atherosclerosis through targetprevent-ing dyslipidemia,5 hypertension,6 and dysregulated
coag-ulation7 have saved lives and reduced systemic
compli-cations, identification of novel targets against disease sequelae is lacking. In addition, there likely remains a substantial group of patients whose susceptibility to atherosclerosis in their peripheral arteries is dispro-portionate to their risk factor burden in other vascu-lar beds and those who continue to rapidly progress
despite best evidence-based management. To address these issues, it is of the utmost importance to develop therapies of direct relevance to patients with PAD, including those with variable responses to therapy or clinical trajectories.
Here, we explore the impact of strategic research col-laboration across the whole translational pipeline to bring new medical solutions for patients with PAD. Building large cohorts of biobanked tissue and blood specimens will provide enhanced opportunity for discovering new mechanisms of disease susceptibility,8 augmented by
high-throughput omics platforms, and massive advances in bioinformatics (Figure 1).9 Ensuring that discoveries
made through such approaches inform in vitro and in vivo models will be key to translating knowledge of disease-promoting pathways into new therapies for patients with vascular disease. We begin by discussing advances and discoveries from such unbiased approaches and oppor-tunities for the future in regard to target identification and prioritization.
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DISCOVERY: TARGET IDENTIFICATION
AND PRIORITIZATION THROUGH
GENOMICS
Unraveling the genetic architecture of vascular disease is fundamental in the effort to innovate and realize novel targets for therapeutic intervention, as well as markers of risk. Translational genomics is perhaps best exemplified by the discovery of PCSK9 (proprotein convertase subtilisin/ kexin type 9) as a molecular target to lower LDL (low-den-sity lipoprotein) cholesterol,10–12 paving the way to explore
alternatives to statin therapy for atherosclerosis, a contin-ued top cause of morbidity and mortality worldwide despite vast efforts to curb its deleterious effects.13,14 As was the
case with PCSK9, uncovering novel therapeutic targets depends upon utilization of next-generation genomics approaches to disentangle disease complexity and home in on key drivers of disease progression. Here, we explore some of the genomics approaches used to study vascular disease in an effort to guide the translational process from target identification to clinical trials (Figure 2).
Genome-Wide Association Studies
Deposition of genetic information into large biobanks and databases has enabled genome-wide association stud-ies (GWAS) that have richly enhanced our understanding of inherited variants which contribute to complex disease phenotypes. Notably, GWAS have highlighted biological processes in the vessel wall and enabled identification of 9p21 as an impactful genetic locus in both coronary artery disease15,16 and PAD.17 Importantly, this locus
con-tains the cyclin-dependent kinase inhibitor CDKN2B (cyclin dependent kinase inhibitor 2B) which was func-tionally shown to be involved in PAD pathogenesis after
Cdkn2b−/− mice developed advanced hindlimb ischemia
(HLI) after femoral artery ligation.18 The Million
Vet-eran Program (MVP),19 a database established in 2011
of genomic data from the Veterans Affairs Healthcare System, is one such archive used to explore PAD sus-ceptibility loci.20 The MVP cohort includes a multiethnic
population that is particularly susceptible to PAD and its comorbid sequelae due to the higher prevalence of smok-ing,21 diabetes, and hypertension,22 making this an ideal
group from which to identify genetic factors that increase PAD risk. Indeed, Klarin et al20 used the MVP data set
and identified 19 total PAD loci that exceeded genome-wide significance. Importantly, their analysis highlighted 11 genetic loci common to coronary, cerebral, and periph-eral vascular disease (LDLR, LPA, LPL, SORT1—lipids;
PTPN11—hypertension; TCF7L2—diabetes), as well as 4
genetic signals specific to PAD, including factor V Leiden variant, F5p.R506Q (thrombosis) and CHRNA3 (smok-ing), suggesting that smoking and thrombosis may play an even greater role in PAD than in other arterial territo-ries. These results also reinforce the use of lipid-lowering therapies to target disease burden across multiple vascu-lar beds and supply the genetic rationale for targeting the coagulation cascade in PAD, as was done in VOYAGER trial (the Vascular Outcomes Study of ASA [acetylsalicylic acid] Along with Rivaroxaban in Endovascular or Surgi-cal Limb Revascularization for PAD [peripheral artery disease]).2 MVP and other large databases are invaluable
resources for assessing novel disease gene variants and potential genetic targets for a range of vascular diseases.
Importantly, while GWAS studies in general may be lim-ited by confounders such as misclassification of case ver-sus control and inappropriate bundling of disease subtypes under a common diagnosis, these issues may be more pronounced in PAD given the fact that many patients with disease have atypical or no symptoms, or because those with confirmed PAD are presumed to have atherosclero-sis when they may, in fact, have some other vasculopathy. In the MVP GWAS, these issues were mitigated by chart review of a representative cohort of cases and controls to verify International Classification of Diseases, Ninth
Revi-sion (ICD-9) coding that correlated with clinician verified
PAD diagnosis (determined through clinic notes and other diagnostic criteria). Improvements in subphenotyping with inclusion of imaging or advanced analysis of plaque charac-terization and disease distribution may help stratify patients in a manner reflecting distinct biology, with the potential to identify loci for specific subtype of PAD that may be diluted by grouping all patients with PAD together.
Next-Generation Omics and Single-Cell
Technology
Implementation of next-generation sequencing has become an important resource in the vascular biologist’s toolkit and may help uncover mechanisms underlying
Nonstandard Abbreviations and Acronyms
3D 3-dimensionalEC endothelial cell
ES embryonic stem
GWAS genome-wide association studies
HLI hindlimb ischemia
ICD-9 International Classification of Diseases, Ninth Revision
iPSC induced pluripotent stem cell
LDL low-density lipoprotein
MVP Million Veteran Program
PAD peripheral arterial disease
PCSK9 proprotein convertase subtilisin/kexin type 9
VEGF vascular endothelial growth factor VSMC vascular smooth muscle cell
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varied response to standard-of-care treatments. For example, although endarterectomy may reduce the risk of stroke in patients with symptomatic stenosis of the internal carotid artery, restenosis has been found in a substantial subset of patients, including over 35% during a 3-year follow-up in one study.23 This may be due to
cel-lular heterogeneity within the plaque or the vascular wall that then influences intercellular behavior and disease progression. Understanding the cell types and differen-tial gene expression patterns found in normal arterial tis-sue versus atherosclerotic plaques could help to refine our understanding of why some patients fail treatments, such as endarterectomy or stenting. Similarly, although antiplatelet therapy is a mainstay for patients with PAD, the prevalence of antiplatelet resistance is comparable to rates seen in cardiovascular disease,24 underscoring the
importance of delineating cellular mechanisms leading to such variations in treatment efficacy.
Bulk sequencing studies, while yielding important over-all genomic insight, obfuscate the differences between cell types contributing to gene expression. Recent efforts have focused on interrogation of the genome and its accessibility at the cell level. Leveraging single-cell technology to examine single-cellular subpopulations and genomic differences presents an important opportunity
to identify disease mechanism and exploit novel targets for therapy. For example, single-cell RNA sequencing has shed critical insight into cardiac vascular differentia-tion,25 endothelial specification,26 clonal smooth muscle
cell (SMC) expansion,27 and hematopoietic potential.28
In a murine HLI model, a small group of fibroblasts that expressed some endothelial cell (EC) genes were further dissected and clustered by single-cell RNA sequenc-ing, revealing subsets of tissue fibroblasts that seem to contribute to angiogenesis under ischemic conditions.29
Many recent efforts have used single-cell RNA sequenc-ing to interrogate the plaque microenvironment due to its complex cellular composition.30–35 We have shown
transcriptional differences in carotid plaque samples between the most advanced portion of the atheroscle-rotic core and a region directly proximal to this location, suggesting a possible anatomic framework for athero-genesis and the possibility of site-specific disruption of disease progression.35 While the majority of these
stud-ies use more easily obtainable tissues, such as plaque from carotid endarterectomy, there is an opportunity to explore other vascular beds subjected to variable hemo-dynamic stressors which may shed light on site-specific genomic differences in plaque burden. For example, atherosclerotic plaque may be obtained from femoral
Figure 1. Schematic demonstrating existing and evolving state-of-the-art components to enhance bench-to-bedside translation in vascular research.
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endarterectomies for dissociation and sequencing, or alternatively full-thickness artery may be harvested from lower extremity revascularization for critical limb-threat-ening ischemia. Transcriptome comparisons may be made between the various vascular beds sampled, lead-ing to potentially novel and significant genomic insight into anatomic disease burden.
Refined examination of intercellular communication and cellular plasticity, both well-appreciated phenomena in vascular biology, further underscores the utility of next-generation sequencing for molecular target identification. For example, vascular SMCs (VSMCs) have been shown to stimulate platelets through CLEC-2 (C-type lectin-like receptor 2), possibly facilitating thrombus formation after plaque erosion.36 Delineating which cell subtypes
are responsible through single-cell analyses may allow for more targeted perturbation of this process, especially as cell-specific nanotherapies evolve.37 Furthermore, the
phenotypic landscape of VSMCs is understood to be quite dynamic as these cells often transition to multiple alter-native phenotypes in the atherosclerotic environment.38,39
VSMCs participate in phenotype switching between con-tractile and synthetic subtypes, and they contribute to the development of extracellular matrix producing cells.39
Advances in sequencing-based lineage tracing may facilitate mapping of clonal histories of VSMCs and other vascular cell types,40 in addition to improved resolution
of clonal substructure as seen in cells within the tumor microenvironment.41 Additionally, many
disease-promot-ing processes are mediated through molecular cross-talk between VSMCs,42 but this type of crosstalk is not
limited to signaling between the same cell types. Ath-erogenesis involves coordinated genomic and molecu-lar communication between the plethora of immune cell subtypes and other cellular components of the arterial wall.43,44 Single-cell technology offers an exciting
oppor-tunity to begin decoding this intercellular communication while also incorporating gene network analysis which, by integration with single-cell RNA sequencing, may shed insight into the underlying biology of groups of cells.35,45
Beyond the transcriptome, detecting chromatin accessibility with single-cell assay for transposase-accessible chromatin-sequencing can unveil layers of biological complexity underlying the transcriptional dif-ferences observed between cells.46 Because the majority
of complex disease genetic variation is noncoding,47 and
functional noncoding gene elements are involved in epi-genetic regulation of gene expression, single-cell assay for transposase-accessible chromatin-sequencing has been used for chromatin-mapping to facilitate analysis of cis- and trans-regulatory elements.48 Integration with
sin-gle-cell RNA sequencing data further allows for refined cell subpopulation analysis through reconstruction of chromatin accessibility profiles of cell types defined by
Figure 2. Opportunities for novel target identification and prioritization in large, well-phenotyped clinical cohorts integrating molecular and histological characterization with genome-wide association studies (GWAS) and single-cell multi-omic technologies.
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RNA profiles.49,50 This has enabled high-resolution
exam-ination of cardiac progenitor cell fates,51 with emerging
opportunity to use this technology to assess chromatin accessibility in the peripheral vasculature.
DNA sequence variations, alternative splicing of mRNA, posttranslational modifications, phosphorylation, and proteolytic cleavage collectively contribute to the diversity of the human proteome far beyond the known 20 000 protein-coding genes.52–54 Cellular indexing of
transcriptomes and epitopes by sequencing allows for simultaneous analysis of the transcriptome and pro-teome via exploitation of DNA barcodes conjugated to
antibodies to enable cell surface protein detection.55,56
It has been used in conjunction with single-cell RNA sequencing for immunophenotyping of carotid plaques to delineate T-cell and macrophage subsets in symptomatic versus asymptomatic carotid plaques.57 Emerging
single-cell mass cytometry methods will allow analysis of pro-teins and their interactions and degradation,58 bringing a
unique tool for evaluation of protein signaling dynamics in the vasculature.
Single-cell analysis of the genome, epigenome, tran-scriptome, and proteome has reshaped the ability to har-ness genetic variation and heterogeneity to identify and
Figure 3. Schematic summary of preclinical cellular and animal models relevant to peripheral arterial disease (PAD). In vitro models include angiogenesis assays and patient-derived cocultured organoids with the opportunity for CRISPR gene editing. In vivo models include hindlimb ischemia and wound healing in mice and abdominal aneurysm model established in pig models.
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exploit novel therapeutic targets. Once identified through genomics approaches, these targets can then undergo functional analysis through in vitro and in vivo model sys-tem assays for feasibility and proof-of-concept before reaching clinical trials.
Multi-Omic Characterization of Large,
Well-Phenotyped Clinical Cohorts to Discover New
Mechanisms and Markers
Understanding the complex interplay between traditional, environmental, and host genome risk factors in deter-mining the susceptibility to PAD requires consideration of biological networks. Tremendous advances have been made in the high-throughput measurement and unbiased
analysis of the metabolome, proteome, lipidome, and the transcriptome.9 However, to make the most of the power
of these technologies, large cohorts with excellent clinical and image phenotyping are required. Application of net-work analytics and machine learning algorithms have the potential to uncover biological signaling pathways and mechanisms that do not rely on prior understanding. From a pragmatic perspective, greater access to tissue and ath-erosclerotic plaque from surgical procedures offers con-siderable advantages compared with similar approaches applied to the coronary or intracerebral disease process. Strategic leadership has led to the establishment of many large collaborative bioresources, including the Munich Vascular Biobank with biomaterial from over 1500 high-grade carotid artery stenoses; >700 PAD and >480 aortic
Figure 4. Schematic clinical trial concept with example intermediate biologically and clinically relevant end points.
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aneurysm cases59; and the Biobank of Karolinska
Endar-terectomies with >200 samples.60 The concomitant
collec-tion of peripheral blood at the time of surgery provides the opportunity to identify a circulating biosignature of particu-lar plaque phenotype and particuparticu-lar vulnerability.61 The UK
Biobank provides additional opportunities based on large scale imaging (100 000 participants) of carotid plaques.62
Such approaches may also unravel some of the mys-teries driving an individual’s differential susceptibility to atherosclerosis in different vascular beds (eg, coronary versus aortic or femoral). Furthermore, the cataloging of samples based on sex and ethnicity, as well as stratifica-tion according to specific phenotypes of atherosclerosis and vascular disease (eg, different patterns of calcifica-tion, tortuosity, morphology, histology, inflammacalcifica-tion, and neovascularization),59 may be combined with multi-omic
analysis in large cohorts may provide insights to thera-peutic targets relevant for specific groups.
The formation of large biorepositories present impor-tant opportunities for precision medicine and genom-ics but include challenges with infrastructure, access to comparison tissue, and sample annotation, all of which are areas of active research for improvement.63
PRECLINICAL VALIDATION:
TRANSLATIONAL MODELS TO UNRAVEL
MECHANISMS AND TEST NEW
THERAPIES
The technologies used to develop new drugs and prod-ucts once definitive targets have been identified are rap-idly expanding, and approaches such as in silico screening, fragment-based drug discovery, and tagged libraries have transformed the pathways available for modern drug dis-covery.64 However, collaborative efforts and strategic
lead-ership are required to ensure that the mechanisms directly relevant to unmet need in human disease are considered in the choice of in vitro and in vivo models to test such com-pounds. This will allow the more efficient and coordinated translation of new, urgently needed therapies (Figure 3).
In Vitro and Ex Vivo Vascular Models
Before advancing to in vivo animal studies, investigators commonly deploy an array of pharmacological studies which rely heavily on in vitro and ex vivo cellular models. This allows for screening of chemical compound libraries and specific novel candidate drugs or products to refine and optimize putative translational targets. Because the patient with PAD commonly experiences ischemia due to insufficient blood flow, a long-standing goal for the vascu-lar research community is to develop therapies that stimu-late the sprouting of new blood vessels (angiogenesis) and trigger the maturation of preexisting collateral net-works (arteriogenesis). There are several well-established
monoculture models that have been used to specifically examine the potential beneficial effects on angiogenesis as it relates to PAD. Here, we discuss both the long-standing monolayer culture approaches, as well as the more recent approaches involving 3-dimensional (3D) spheroids, coculture, and microphysiological systems.
In vitro functional assays provide a valuable tool for assessing the effect of angiogenesis. They use basic cell culture techniques which can be quickly and easily per-formed and analyzed quantitatively. They are essential for investigating the biochemical, cellular, and molecular mechanisms occurring in angiogenesis because individual components of angiogenesis can be assessed in isolation. ECs undergo multiple processes during angiogenesis, including cell proliferation, migration, sprouting, branching, and tubule formation, as well as complex cell-cell interac-tions. In addition, ECs differ depending on their vascular bed and vessel type—the most commonly used EC type in angiogenic assays include HUVECs (human umbilical vein endothelial cells), HAECs (human aortic endothelial cells), and HMECs (human microvascular endothelial cells).65
Proliferation
The process of EC proliferation can be quantified using several different methods in vitro. The simplest method for assessing EC proliferation is counting cells. This can be achieved using the common hemocytometer and a viabil-ity dye, such as trypan blue, or through the use of auto-mated cell counters. Colorimetric assays like the MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-Diphenyltetrazolium Bromide) or the WST-1 (4-[3-(4-Iodophenyl)-2-(4-nitro-phenyl)-2H-5-tetrazolio]-1,3-benzene sulfonate) assay are widely used to assess the natural byproduct of cellular division.66 Essentially these assays determine cell viability
more so than proliferation,67 however, they are commonly
used as methods to quantify EC proliferation in angiogen-esis. Another potentially more accurate method used to assess proliferation includes quantifying DNA synthesis via BrdU (bromodeoxyuridine / 5-bromo-2'-deoxyuridine) incorporation68 or Click-iT via EdU incorporation.69
Migration
After proliferation, ECs migrate away from their origin. All angiogenic models involve the sprouting of ECs from a monolayer, and the movement of ECs through the base-ment membrane is often referred to as sprouting and occurs due to nearby chemical signals that either promote or prevent angiogenic activity. Assessing the migratory ability of ECs can be achieved with a number of differ-ent assays each with their own advantages and disadvan-tages. Wound healing or the scratch assay is the simplest and quickest assay to perform. ECs grown to a conflu-ent monolayer are wounded commonly with a pipet tip or other similar hard objects to create a denuded zone.70
In response to this damage, ECs begin to proliferate and migrate into the denuded zone to heal the wound. The clear disadvantage of this assay is the reproducibility with
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UM issues in confluency of cells, consistency of the scratch, and difficulty in accurate quantitation.70 Transfilter assays
or Boyden chamber assays are generally used to study migration of ECs towards a stimulus (commonly VEGF [vascular endothelial growth factor]) and have the advan-tage of distinguishing between specific directional migra-tion instead of random cell movement.68 ECs are seeded
into the upper chamber with the stimulus in the lower chamber in culture medium. The migration of ECs through the filter can take anywhere from several hours to 48 hours and can be quantified by cell staining or counting of cells attached to the filter. The main weakness of this assay is the inability to observe real-time migration. New automated technologies are now able to overcome this limitation with the use of systems such as xCELLigence (Roche)71 and also the electric cell-substrate impedance
sensing instrument (Applied Biophysics).72 These systems
allow real-time monitoring of cell proliferation and migra-tion when cells are grown in special electrode-covered chambers. The resistance generated by the cell mem-brane increases the electrical potential between the elec-trodes, and this allows monitoring of the cell behavior.65
Tube Formation
Tubulogenesis, more commonly termed tube formation, is the connection of the new blood vessels to preexisting vessels beds. Modeling this process in vitro is a corner-stone of angiogenic research. Using ECs grown in cul-ture and then placed on commercially available Matrigel, this assay is a 2-dimensional imitation of extracellular matrix. The Matrigel acts as the extracellular matrix con-taining cytokines and growth factors allowing the ECs to form tubule-like structures.66 Tube formation can be
observed with imaging techniques and can take any-where from 3 to 24 hours to provide reliable data. The results obtained can be highly variable and comparisons in the literature depend widely on cell type, quantity of Matrigel used, and end point analyzed (tubule number, tubule length, or branch points).68 Furthermore,
differ-ent methods of analysis allow for increased variability, whether these data points are measured manually, which is more arduous and time consuming, or using auto-mated image analysis programs, which require screen-ing to eliminate artifacts but can quantify at a faster rate. This 2-dimensional model has since been expanded to 3D assays to more closely mimic the angiogenic sprout-ing of ECs observed in vivo.68 Simple conversions of this
assay include using thicker basement matrix so ECs can be seeded at differing levels to observe tubule formation both vertically and horizontally. More complex versions include scaffold free cell sheet stacking, cell aggregate microtissue assays, and microcarrier spherical scaffold assays.65,73 All of these have the advantage of
recapitu-lating all the stages of in vivo angiogenesis, however, the technology required for visualization and analysis can be costly and difficult to interpret. 3D models have also expanded to include coculture incorporating not just
ECs but also vascular SMCs and fibroblasts.18,74 Such
approaches may enhance the modeling of in vivo human disease and have the capacity to support early drug screening, potentially reducing the number of animal studies required. However, it is essential we improve the uniformity of organoid models at the necessary scale. Proof of translatability is still a major issue, similar to all preclinical models in the PAD space.
Embryoid Body Vasculogenesis Assay
Embryonic stem cells (ES cells) have been highlighted as promising cell sources for the study of differentiation towards the endothelial lineage and vasculogenesis75–77
since they can go through the majority stages of angiogen-esis observed in vivo.78 This assay requires ES cells to be
initially differentiated into embryoid bodies, which are then placed into a 3D collagen matrix for the embryoid bod-ies primary vascular structures to extend and invade the collagen matrix, leading to sprouting of ECs similar to the angiogenic process.79 Using cell imaging, this assay allows
for qualitative and quantitative analysis of multiple steps in angiogenesis and makes it possible to test many different therapeutic agents.80 While the assay is comprehensive, it
is time consuming both at the experimental and analyti-cal stages.81 Additionally, the use of ES cells in biomedical
research has been hindered by ethical issues since extrac-tion of these cells disrupts the human embryo.82,83 Even
with several studies demonstrating effective use of these cells in the study of angiogenesis, ES cells are not gener-ally considered an ethical source of ECs.79
Induced Pluripotent Stem Cells-Disease in a Dish Overcoming the ethical issues with the use of ES cells, a breakthrough discovery in cellular biology was the dis-covery of induced pluripotent stem cells (iPSC) made by Takahashi in 2006.84 With advances in cellular
repro-gramming, iPSCs are derived from adult somatic cells85
and blood,86,87 into a stem cell-like state by the
introduc-tion of the common transcripintroduc-tion factors OCT4 (octamer-binding transcription factor 4), SOX2 (sex determining region Y-box 2), KLF4 (kruppel-like factor 4), and c-MYC (cellular Myelocytomatosis) (Yamanaka factors) via viral transduction, protein and microRNA transduction, or by chemical/small molecule-based reprogramming strate-gies.88 The first use of iPSC in the field of vascular
biol-ogy occurred in 2009, when Taura et al89 demonstrated
the ability to yield ECs from iPSC (iPSC-EC), which when matured display phenotypic properties highly similar to primary ECs.90 More recently, iPSC-ECs have shown
therapeutic potential by enhancing angiogenesis in mouse models with improvements in blood reperfusion and enhanced wound closure.91,92 A major advantage of
iPSCs is that they can be easily generated from patients with or without disease for use in angiogenic assays.93
Blood vessel development can be modeled in 3D in vitro assays using iPSC-ECs cultured within engineered plat-forms that mimic the 3D microenvironment.94 Hydrogel
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scaffolds, either on standard well plates or within a pas-sive pumping microfluidic device, have offered a more defined platform for vascular-based assays.95,96 Major
advances in genome editing techniques with the use of clustered regularly interspaced palindromic repeats/ Cas9 allows causality of genetic variants to be explored in a rigorous manner in patient-derived iPSCs and derived vascular cells97 with full characterization of cell
signaling and molecular profiling. As the application of iPSC-derived vascular cells and models evolve, it will be critical for the field to develop standardized protocols.85,98
Aortic Ring Assays
Beyond the standard assays outlined above, investigators commonly employ organ explant assays, which appear to synergistically assess almost all steps of physiological angiogenesis.99 Aortic ring assays, often termed ex vivo
models, have been developed to be the in vitro mimic of in vivo angiogenesis with the ability to measure angio-genic sprouting, outward growth, and (to an extent) sta-bilization of new blood vessels from explanted segments of vasculature.100,101 Vessel rings are excised, cleaned,
cut into ≈1 mm sections, immersed in collagen, fibrin, or Matrigel and cultured for vessel outgrowth. After a few days, the outgrowth can be measured using staining and microscopy, then compared with samples that have been exposed to potential angiogenic agents. The advantages of the aortic ring assay are numerous including the cap-ture of multiple cell organization with additional paracrine support and extracellular matrix components.68,99 The
downside of this assay is reproducibility due to imprecise segments and the absence of blood flow effects.
Vascular Organoids (Scaffold Free)
The assembly of different cell types into 3D spheroid cocultures called vascular organoids are being described as a bridge in the gap between traditional 2-dimensional in vitro cell culture and the complexity of animal mod-els. These transformational 3D model systems provide a more physiologically relevant assay encompassing complex cell-cell interactions, which enable the deposi-tion of their own extracellular matrix.102,103 The advent
of hybrid spheroids using more than one cell type pro-vide useful models for angiogenesis assays with the formation of tubular-like structures.104,105 Multiple
dif-ferent techniques, including the hanging-drop, the use of low adhesion plates, or self-organizing vessel-like structures, have been used to assess angiogenesis. While these methods can be relatively cost-effective, they are time consuming and require advanced imaging capabilities for analysis.106 More recently, these
meth-ods use the addition of iPSC-ECs with multiple other cell types to increase the ability for cells to reorganize and form complex vascularized networks.107,108 Studies
have shown that after implantation of iPSC-EC organ-oids into the chick chorioallantoic membrane assay, the generated vessels connect with the host circulation and
vessel structure is preserved.108 Many aspects of vessel
formation within the vascular organoid can be detected including branching, an extracellular matrix, cell-cell interaction, microvesicle release and responsiveness to proangiogenic and antiangiogenic stimuli.109
Vascular Scaffolds
In addition to the more commonly used scaffold of Matri-gel to assess EC interactions in angiogenic-based assays, other more complex vascular networks are being bioengi-neered to incorporate the missing in vivo elements. Elec-trospinning and 3D bioprinting are being used to address the need for a more complete microenvironment includ-ing cell organization, spatial control, and localized release of growth factors in response to cues from the extracel-lular matrix and localized blood flow. Electrospinning can produce nanofiber-based synthetic vascular networks with similar dimensions to naturally occurring extracellular matrix (50–500 nm) and provides an excellent platform for the adhesion of ECs in a monolayer. A limitation in this methodology, however, is that nanofibers create a physi-cal barrier for cells, and this limits the ability to mimic the permeability of the endothelial monolayer.110 3D
bioprint-ing is an emergbioprint-ing approach to generate scaffolds with hydrogel, which can be embedded with microchannels to study angiogenesis.111,112 Using either direct or indirect
printing methods, cells, biomaterials, and growth factors can be combined to produce complex shaped constructs with defined micron-sized channels and pore sizes that are capable of guiding angiogenesis. Both electrospin-ning and 3D printing techniques have various advantages and are frequently used in tissue engineering applica-tions. The 3D bioprinting technique allows controlling the production of a large number of scaffolds with precise measurements relatively quickly while electrospinning builds scaffolds with a wide range of properties to mimic blood vessel structure including composition, diameter, thickness, porosity, and degradation rates.112
Nanoparticles
The testing of targeted angiogenic pathways has been significantly enhanced by technological advances in nanotechnology. More efficacious administration of angiogenesis modulators using nanoparticles, includ-ing metallic, metal oxide, glass-ceramic, and polymeric nanoparticles, has allowed for treatments with a lon-ger half-life and more selective targeting of the vas-culature.113–116 Some of the first studies used albumin
and gelatin nanoparticles containing a DNA plasmid to trap excess VEGF and reduce angiogenesis,117 while
alternative applications were designed to increase angiogenesis and enhance revascularization.117–119
Clinical trials involving VEGF therapy have been unsuccessful at improving peak walking time, ankle-brachial index, or quality of life,120,121 thus
underscor-ing the need to evaluate each step of the translational cascade described.
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UM tubes containing microRNA oligonucleotides and VEGF-Other platforms include single- and multi-walled nano-targeted siRNA, able to regulate EC proliferation and prevent angiogenesis.122,123 Graphene oxide nanoparticles
structures have demonstrated increased proliferation and migration of cells to promote wound healing and nano dia-monds can increase bioavailability of angiogenic drugs for improved delivery to tissue.124,125 Among metallic options,
gold nanoparticles are being recognized for their angiogenic potential.116 They are composed of an inorganic core
con-taining gold encircled by an organic monolayer and have displayed numerous antiangiogenic effects by suppress-ing activation of VEGFR2, Tie2R (TEK receptor tyrosine kinase 2), FGFR (fibroblast growth factor receptor), and many of their downstream signaling pathways.113,126,127 While
the majority of these applications have been targeted for tumor-related angiogenesis and, therefore, focus on antian-giogenic properties, there are some applications that have demonstrated proangiogenic effects,128 with increased cell
survival and proliferation of ECs and increased vessel-like structures, particularly in wound healing.129,130 Nanoparticle
characterization is increasingly variable and, therefore, dif-ficult to compare across multiple studies. Given the missing details in some publications, there has been a call for mini-mum information reporting with regards to the details in the study of nanomaterials and their biological interactions.131
Obtaining a standard in the field will improve the growth of available and effective nanoparticles for use in angiogenesis.
Specific vascular targeting nanoparticles continue to be developed in an effort to advance precision ther-apy for atherosclerosis. Lesional macrophage-specific single-walled nanotubes are shown to accumulate in the atherosclerotic plaque and, with the appropriately coupled therapeutic, reactivate efferocytosis to reduce plaque burden with minimal off-target cytotoxicity.37
Targeting of vascular inflammation using leukosomes, a biomimetic nanoparticle that integrates leukocyte-derived membrane proteins into the phospholipid bilayer, has been shown to enhance delivery of therapeutic to inflamed endothelium at sites of plaque development.132
Continued efforts to refine cell type-specific therapeutic delivery will likely be critical for future vascular therapies.
Preclinical In Vivo Models
Once a novel target gene or therapeutic candidate has been identified, the preceding in vitro assays are com-monly used to determine which leads should be priori-tized for additional in vivo testing. In the field of vascular medicine, investigators tend to be broadly focused on determining whether a given pathway may regulate new blood vessel formation, peripheral atherosclerosis, isch-emic tissue wound healing, and thrombosis.133 In the
following section, we provide a general overview of the in vivo mouse models available to test these processes, including the strengths and limitations of each approach,
as well as emerging techniques that may allow more sophisticated analyses of the biology underlying periph-eral vascular disease.
HLI Models
As discussed previously, mitigation of ischemic com-plications in PAD through stimulation of angiogenesis and arteriogenesis are long-standing goals in vascular research. These processes can be assessed several ways (including with retinal angiogenesis or Matrigel implanta-tion assays) but are most commonly tested in the murine HLI model.134,135 This model relies on the interruption of
lower extremity blood flow, most often induced by the ligation of the femoral artery (which can model claudica-tion given that collaterals downstream of the profunda femoral artery are left intact) or via the simultaneous liga-tion and excision of the femoral artery (which can model critical limb ischemia given that the collateral circulation is also perturbed with this approach). Limb perfusion is then quantified by noninvasive laser Doppler imaging, where the flux of blood (normalized to the nonischemic limb) can be quantified during recovery or in response to therapeu-tic intervention. Other reproducible readouts include the use of microcomputed tomography angiography, digital necrosis scoring, and blinded histological quantification of neovessel density.18 Studies in large animal models of
PAD have had success in mimicking the ischemic myopa-thy seen in ischemic human limbs by using endovascular techniques and occluding inline flow through the external iliac artery and impeding the rapid arterial collateralization through the use of a covered stent.136
While the HLI model is commonly used, it does have several limitations that warrant discussion.135 First, these
studies are most often performed in young, nonathero-sclerotic mice without any of the comorbidities typically encountered in humans with PAD. While some have advocated for the use of diabetic (eg, db/db) or dyslipid-emic (eg, apoE−/−) animals, it is important to note that the
acute cessation of flow in an otherwise healthy artery is perhaps more reflective of acute limb ischemia than the gradual reduction in perfusion that is observed in vascu-lopathy who experience reductions in their ankle-brachial indices over a period of months to years. Models that use an ameroid constrictor device (which can gradually occlude the vessel) have been reported and are being studied as a potentially more relevant simulator of human vascular disease.137 Second, it is critical to recognize that
there are profound differences across mouse strains which can influence the interpretation of results with this model.138 For example, the widely used C57Bl/6 strain
has a robust collateral reserve and is resistant to critical limb ischemia, while the BALB/c strain is highly suscep-tible to digital necrosis and even limb loss after femoral ligation. Thus, investigators must consider whether they are interested in modeling claudication or CLI before
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generating a new genetic knockout model or embarking on a therapeutic intervention study. While multiple modal-ities can be used to quantify limb perfusion, each has its own intrinsic limitations, as previously summarized by Lotfi et al.135 In the particular case of laser Doppler
imag-ing, factors that can alter vasoconstriction or vasomotor tone (eg, depth of sedation, room temperature, animal sex, etc) should be controlled to the best extent possi-ble.139 Finally, studies exploring the cell-cell interactions
occurring during limb perfusion are providing mechanis-tic insights into vascular repair. More than simply an EC proliferative response, the involvement of VSMCs, mac-rophages, and pericytes are contributing at a molecular level to participate in tissue perfusion.
Peripheral Plaque Vulnerability Models
Given that most PAD is atherothrombotic in nature,140 the
translational vascular research community also uses a wide array of mouse models that develop atherosclerotic plaques in the peripheral vasculature, including the aorta and brachiocephalic artery. Traditional dyslipidemia mod-els (eg, LDLR−/− and apoE−/− mice fed a high fat Western
diet) can be used to reproducibly quantify plaque burden (using lipid stains such as oil red O) and lesion composi-tion (with histological analysis of necrotic core size, foam cell burden, and fibrous cap thickness). However, most commonly used murine atherosclerosis models are not susceptible to spontaneous plaque rupture and do not consistently demonstrate the intraplaque hemorrhage and superimposed luminal thrombosis encountered in subjects with PAD. Accordingly, a variety of models that induce mechanical alterations in blood flow to promote endothelial dysfunction and plaque destabilization have now been described. These methods (which include inter-position grafting, perivascular device placement, arterial ligations, or fistula formation) were recently reviewed by Winkel et al,141 including their predicted impact on shear
stress and how those changes can be used to model various aspects of peripheral vascular disease.142
Amongst these models, the application of an extrinsic silastic collar to the carotid artery (with143 or without144,145
angiotensin infusion) seems to have emerged as the approach which most faithfully recapitulates the human condition.146 While technical variations in the approach
exist,147 these so-called cuff or cast models promote
oscil-latory and reduced shear stress in the branch vessels of the aortic arch, thus leading to the generation of advanced and destabilized carotid lesions. These plaques demon-strate a high burden of macrophage infiltrates, a reduction in collagen and SMC content, thinning of the fibrous cap, and intraplaque hemorrhage.147 Such vulnerability models
can be deployed in mice with indelible cell-specific lineage tracers, which are recognized as being highly informa-tive given the increasingly appreciated role for phenotype switching, cellular plasticity, and clonal expansion during
atherogenesis.27,148,149 Moreover, our group recently
demon-strated that cast-induced changes in lesion vulnerability can be quantified noninvasively (using 18F-FDG-PET (positron
emission tomography with 2-deoxy-2-fluorine-18-fluoro-D-glucose) computed tomography scanning) and that this imaging modality is sensitive enough to detect changes in plaque stability after drug treatment over time.150
Accord-ingly, these peripheral plaque rupture models may prove useful for investigators studying new translational therapies.
Ischemic Wound Healing Models
Due to poor circulation and prolonged tissue ischemia, many patients with PAD develop chronic wounds or ulcers on their lower limbs. These can be very difficult to heal and often become infected and require amputation of the affected foot or leg.151,152 In addition to the health burden,
the economic consequences are substantial. The com-plications associated with infection and amputation also lead to increased mortality in these patients, particularly in patients with diabetes.151 Wound healing is a process not
able to be replicated in cell models and has primarily been limited to large animal models, such as swine, to ensure a comparable process to humans.153 In the past 2 decades,
however, murine in vivo wound healing experiments have become effective models for studying human wound heal-ing.154 Three reproducible murine wound healing models
that recapitulate the human wound healing process have been described—the splinted excisional wound,155
isch-emia-reperfusion model,156 and the ischemic flap model.154
Excisional wounds on the dorsal surface are the most com-monly used wound healing model, however, in mice wound contraction is a significant limitation in assessing wound clo-sure. The use of splints, therefore, increases the relevancy of this model to human wound healing.157 Silicone splints
are fixed to the wounds to prevent contraction and allow the wound to heal through tissue formation and reepithelializa-tion.153,155 Despite this, it still remains a great challenge to
replicate wound healing as murine models present different anatomy to humans, particularly for the use of rodent skin. In a recent systematic review,158 animal models of ischemic
wound healing were investigated with only 3 studies using more clinically relevant wounds created on the paws of mice after undergoing HLI.159–161 Some recommendations
to help improve this model includes the use of aged mice to mimic human diseases, improving bias, and standardizing assessments for the ideal ischemic ulcer model in addition to using a more stable induction of ischemia.137
FROM BENCH-TO-BEDSIDE:
ACCELERATING TRANSLATION THROUGH
EARLY PHASE CLINICAL TRIALS
While there have been substantial improvements in preclinical models of PAD, the disease complexity has
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UM contributed to disappointing results in the clinical trans-lation of therapies showing potential at earlier stages. Collaboration at the interface of the preclinical and clini-cal efforts is required to improve the prioritization of the most promising novel therapies and to strategize how to overcome developmental hurdles. More efficient early phase studies focused on biologically relevant end points in more precisely stratified patients will be key. In addi-tion to tradiaddi-tional and novel systemically administered therapies, the peripheral and accessible nature of PAD should also make innovative approaches to local deliv-ery of novel therapies feasible. This may involve stents or bioresorbable scaffolds, or topical nanoparticle delivery, for example, to ischemic ulcers. Outlined below are con-cepts that may be useful in accelerating the translation of discoveries from bench-to-bedside (Figure 4).
Precision Stratification of Patients Based on
Biology
There remains a considerable degree of disease hetero-geneity in PAD that directly relates to underlying biologi-cal mechanisms, as outlined above. A key consequence of such heterogeneity is dilution of the average therapeutic effect in trials, thus necessitating larger, longer, and more expensive clinical development programs. Approaches described in this review, including genomics, molecular phenotyping, and improved imaging techniques, will pro-vide new risk stratification tools with better opportunity to divide by biology. While pursuing broadly generalizable therapies with that can be used by all vascular patients obviously remains a key goal, testing new treatments in the groups where the therapy makes the most biological sense may help reduce the size of studies and facilitate the demonstration of the impact of the intervention.
Identification of Those With Greatest Unmet
Need
A current unmet need in the management of cardio-vascular disease is the ability to address the substantial number of individuals who develop disease or progress despite best practice treatment. While there is an obvi-ous group of patients who develop disease due to a his-tory of smoking and mixed adherence to recommended treatments, there are also many patients whose disease is driven by unknown biological susceptibility. While at a community and policy level it makes sense to invest in the widespread use of cost-effective and evidence-based care, recognizing that there are subgroups of individuals who demonstrate rapid disease progression, similar to cancer patients with metastasis on therapy, is key. Emerging machine learning and neural networking approaches are beginning to allow for highly accurate subphenotyping of those most susceptible to disease or those least likely to respond to conventional therapy.162
These clinical and genetic enrichment-based approaches will enhance discovery of novel biological mechanisms that are not addressed by existing agents, as well as improve our ability to rapidly test new therapies in early phase trials due to higher event rates and smaller sample size requirements.
Enhancing Our Focus on Biologically Relevant
Intermediate End Points
Efficient translational pipelines require consideration of staged regulatory approaches. Vascular research has set itself an extremely high bar, often using major adverse cardiovascular events or major adverse limb events as the primary end points in late-stage clini-cal trials. Using traditional study design, and minimally stratified recruitment approaches, this high bar drives the need for massive recruitment requirements and high study costs. As a result, many important trials are slower than expected, fail to fully enroll, or run out of funding while waiting for events to accrue.163 Working with
regu-latory authorities to derive and agree upon biologically meaningful intermediate end points with a direct role in disease progression and patient-reported outcomes is, therefore, of critical importance. In the case of oncology, tumor size and its impact on quality of life are relatively obvious measures. In the case of PAD, consideration of vascular function, tissue perfusion, lesion vulnerability, or degree of plaque inflammation may all be considered. In addition, rigorous definition and consensus around tissue-specific end points may be a unique opportunity for PAD within the cardiovascular field. This relates to our ability to access relevant tissue much more easily than cardiologists- such as at the time of carotid endar-terectomy, debridement, or amputation. The field could consider more strategic ways of using this in design of clinical trials unlocking the possibility of novel biologi-cally relevant intermediate end points for new therapies. Further opportunities for biologically relevant end points with potential for mechanistic insights include measures of vascular function (eg, EndoPAT164) or vascular
inflam-mation and plaque burden (FDG/PET computed tomog-raphy165). Remembering our goal of not only helping
patients live longer but also feel better, disease-specific end points of relevance to the patient with PAD should also be more actively integrated into clinical develop-ment programs, including improvedevelop-ments in claudication symptoms or the ability to walk more freely at home (quantified via mobile health devices).
FUTURE DIRECTIONS
Vascular biology research continues to build upon important discoveries by embracing the translational approaches described in this review. Next-generation genomics should not be considered an adjunct but
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rather a critical component of vascular studies used to elucidate mechanisms of disease at the cell-specific level. Delineation of disease process in immune cells versus cells of the vascular wall, for example, may inform future therapeutic direction. Correlation and integration of this data with population-wide GWAS studies may form the foundation upon which novel ther-apies are pursued in clinical trials, which clearly can be enhanced and streamlined. It is also critical to use and improve upon existing animal models of disease. Com-bining animal models, sophisticated in vitro techniques, and advanced genomics will ultimately bring impor-tant discoveries in vascular research closer to trans-lational potential. Consideration and consensus on the use of new biologically meaningful intermediate end points in PAD disease progression will be crucial for future clinical trials. Finally, as investigators continue to make discoveries, new challenges emerge about how discovery turns into useful therapy for clinical prac-tice. One consideration is that while the vasculature serves as a conduit for systemic therapeutic delivery, how are blood vessels themselves targeted in a spe-cific and meaningful manner? Based on the disease process being studied, investigators should continue to assess whether targeting disease systemically or at its nidus would provide the most benefit. Along these lines, refinement of tissue and cell-specific therapeu-tic delivery systems will impact vascular research that relies on a targeted approach for disease amelioration. Collectively, the challenges and opportunities outlined in this review ensure an exciting future for translational vascular biology, hopefully bringing enhanced vascular care closer to patients who increasingly need it.
ARTICLE INFORMATION
Affiliations
Division of Vascular Surgery, Department of Surgery, Stanford University School of Medicine, CA (T.A., N.J.L.). Cardiothoracic and Vascular Health, Kolling In-stitute and Department of Cardiology, Royal North Shore Hospital, Northern Sydney Local Health District, Australia (B.A.D.B., G.A.F.). Centenary Institute, Sydney, Australia (J.M.).
Sources of Funding
G.A. Figtree is supported by a National Health and Medical Research Council Practitioner Fellowship (grant number APP11359290), Heart Research Austra-lia, and the New South Wales Office of Health and Medical Research. This review was also supported by the National Institutes of Health (NIH) R35 HL144475 awarded to N.J. Leeper and the Ansell Fellowship awarded to T. Alsaigh.
Disclosures
G.A. Figtree reports personal consulting fees from CSL and grants from Abbott Diagnostic outside the submitted work. In addition, G.A. Figtree has a patent Bio-markers, and Oxidative Stress awarded USA May 2017 (US9638699B2) issued to Northern Sydney Local Health District. N.J. Leeper reports personal consulting fees from Janssen. The other author report no conflicts.
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