Yearbook of Vascular and Endovascular Surgery 2016 R Sekhar
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Advances in Translational Vascular ResearchCHAPTER 1

J De Siqueira,
G Atturu,
S Homer-Vanniasinkam
 
INTRODUCTION
Cardiovascular science is a broad and complex field ranging from the interaction of microRNA molecules in the cell nucleus to the clinical evaluation of large vessel grafts and stents. Given the breadth of practice of a modern vascular surgeon (arterial, venous and lymphatic pathology; aneurysmal and occlusive disease; ulcer management; medical, endovascular and open surgical treatment), it is indeed a challenge to cover the entire topic in a single chapter.
We have chosen to focus on some of the more recent advances in research in arterial pathology which should be of interest to the general vascular surgeon: regression of atherosclerosis, angiogenesis, chronic foot ulcers and the modulation of aneurysmal dilatation. The advances in translational research described in this chapter have the potential to impact future management strategies for the prevention and treatment of vascular disease.
 
REGRESSION OF ATHEROSCLEROSIS
Atherosclerotic cardiovascular disease remains the leading cause of morbidity and mortality in most countries. Atherogenesis, the process of plaque formation, is complex and involves systemic and local factors. It generally runs a benign indolent course until the plaque ruptures, when the patient presents with acute symptoms.1 Currently, research efforts in this area focus on (i) identifying the vulnerable plaque2,3 in an attempt to prevent plaque rupture and thrombosis, and (ii) atherosclerosis regression. Achieving atherosclerosis regression, although it seems implausible, has been at the centre of research efforts for approximately 60 years.4 In translational clinical studies, both the lipid theory5 and the inflammation theory6 of atherosclerosis have been put to the test.
Dyslipidaemia is a well-known modifiable risk factor for atherosclerosis. Lowering the total plasma cholesterol level using statin therapy has been shown to reduce the incidence of fatal and non-fatal cardiovascular events by 225%.7 Currently, there are approximately 24 different antilipidaemic drugs on the market with five different modes of action.8 The commonly used class of drugs are the statins, which act on the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-Co-Reductase inhibitors). Other drugs act on different pathways of lipid metabolism such as: inhibiting intestinal cholesterol absorption (ezetimibe), increasing hepatic bile acid synthesis (bile acid sequestrants – cholestyramine) and activation of peroxisome proliferator activator receptor α (PPAR α – the fibrates). Vitamin B3 (nicotinic acid or niacin) is a potent lipid-modifying drug that acts on multiple targets. In spite of the availability of a large number of antilipidaemic drugs, this therapeutic modality achieves only about 30% cardiovascular risk reduction in clinical practice. Efforts in this area continue, with over 50 novel drug candidates in the development pipeline, aimed at different molecular targets. Current antilipidaemic drugs are orally active small organic molecules, in contrast to several of the newer agents which are biological products ranging from monoclonal antibodies to gene therapy, requiring invasive routes of administration.
One of the commonest target molecules for these newer generation drugs is proprotein convertase subtilisin/kexin type 9 (PCSK9). PCSK9 is a serine protease synthesised by the liver and intestine, which binds to the LDL receptor and promotes its degradation. Heterozygous loss-of-function mutation of PCSK9 is seen in around 3% of Afro-Americans and is associated with hypocholesterolaemia and an 88% reduction in cardiovascular risk.9 Alirocumab and evolocumab are two monoclonal anti-PCSK9 antibodies that have successfully completed Phase 3 trials and are awaiting approval. In the ODYSSEY trial, addition of 150 mg of alirocumab, administered subcutaneously every fortnight with a statin, was shown to reduce the low-density lipoprotein (LDL)-C by 61% from baseline, compared to a placebo which caused a reduction of 0.8%, with a 54% risk reduction in the absolute rate of cardiovascular events.10 In the DESCARTES study, which is a multicentre, randomised double-blind, placebo-controlled Phase 3 trial, addition of 420 mg of evolocumab (subcutaneously every 4 weeks) to other lipid-modifiying therapies, such as diet control, low-dose and high-dose atorvastatin and ezetimibe, achieved an additional reduction of 48.5–61.2% in low-density lipoprotein (LDL) cholesterol.11
A further area of lipid research interest focuses on increasing high-density lipoprotein (HDL-C) levels by the development of inhibitors of cholesterol ester transfer protein (CETP).12 CETP catalyses the transfer of cholesterol ester from HDL particles to LDL and VLDL (very low-density lipoprotein) particles. Anacetrapib, a potent HDL-C increasing drug, when used at a dose of 100 mg per day in addition to a statin, has been shown to increase the HDL-C by 138% and decrease LDL-C by 40% without significant toxicity, in a multicentre double-blind placebo-controlled Phase 3 trial (DEFINE trial).13 Evacetrapib is another potent inhibitor of CETP that is proven to increase HDL-C levels in a randomised control trial.14 However, both these drugs, in addition to increasing the HDL-C levels also reduce the plasma LDL-C levels, making it difficult to differentiate the specific effect of increased HDL-C levels in reducing cardiovascular risk.
3Gene modulation using antisense oligonucleotide or ribonucleic acid (RNA)-silencing technologies is a powerful way of inhibiting specific target proteins.15 Mipomersen (KYNAMRO®) is the first FDA-approved anti-apolipoprotein B (ApoB) antisense oligonucleotide for treating patients with familial hypercholesterolaemia. Volanesorsen is another apolipoprotein CIII (ApoCIII) antisense oligonucleotide that has reached Phase 3 clinical trials. The results of the two large placebo-controlled randomised trials, APPROACH and BROADEN, are awaited with interest.
In contrast to the lipid-modifying drug trials, those exploring the effect of anti-inflammatory properties in reducing cardiovascular events have met with little success.16 Highly-sensitive C-reactive protein (hs-CRP), a biomarker of inflammation, is shown to independently predict the risk of cardiovascular disease. Clinical studies such as CARE and JUPITER17 have shown that statins reduce both the LDL-C and CRP levels; however, it was difficult to attribute the individual contribution of these two effects to the overall reduction in cardiovascular events.
Peroxisome proliferator-activated receptor-γ (PPARγ) factor is shown to affect glucose metabolism and vascular inflammation. Two PPARγ agonists (also known as insulin sensitisers), rosiglitazone and pioglitazone, were studied in large randomised controlled trials. In the RECORD trial, rosiglitazone did not show any significant reduction in cardiovascular events.18 In the PROactive study, pioglitazone was shown to significantly reduce the occurrence of fatal and non-fatal myocardial infarction.19
Interleukins (IL), mainly IL-1β and IL-6, are other targets of anti-inflammatory clinical trials in atherosclerosis. Canakinumab is a monoclonal antibody against IL-1β. The efficacy of canakinumab in reducing cardiovascular events is the subject of a large study, the Canakinumab Anti-inflammatory Thrombosis Outcome Study (CANTOS), involving 17,200 patients.20 Similarly, the efficacy in reducing cardiovascular events of low-dose methotrexate, by its effect on the IL-6 pathway, is being studied in the Cardiovascular Inflammation Reduction Trial (CIRT) involving 7,000 patients.21 The results from both these studies are awaited.
 
ANGIOGENESIS
Therapeutic angiogenesis is an exciting field of translational research for clinicians who manage patients with occlusive arterial disease. As evidenced by serial imaging, patients on best medical therapy are able to bypass occlusive or stenotic disease by vessel proliferation. The attempt to reproduce or accelerate this process is called therapeutic angiogenesis, which can be brought about by the delivery of angiogenic factors (growth factors, cytokines) locally or by inducing cells in ischaemic tissues to secrete the proteins of interest (gene therapy). Gene therapy carries an inherent advantage over protein delivery – if cells can be programmed to secrete a protein of interest continuously, its delivery can be sustained more easily than with multiple administrations.22 From evidence gathered in pre-clinical studies, five angiogenic factors have been investigated in clinical trials of peripheral arterial disease.
4Vascular endothelial growth factor (VEGF) promotes endothelial cell migration, proliferation and angiogenesis. Although initial small studies showed promising results with angiographic evidence of increased vascularity, the Phase 2 randomised controlled trial using VEGF121 (RAVE trial) in patients with disabling intermittent claudication did not show significant changes in peak walking time or ankle brachial pressure index (ABPI).23 In a more recent randomised clinical trial, intramuscular injection of VEGF165 gene in patients with diabetes mellitus and critical limb ischaemia (CLI) showed improvements in healing of skin ulceration, ABPI and toe brachial pressure index (TBPI) without significant changes in amputation rates or rest pain.24
Fibroblast growth factor (FGF) is another angiogenic factor that enhances blood vessel formation. The initial Phase 2 TALISMAN trial was encouraging with decreased amputation rates.25 However, these results were not reproduced in the follow-up Phase 3 TAMARIS trial.26
Hepatocyte growth factor (HGF), in addition to promoting angiogenesis, has an established role in myogenesis and wound-healing. It is also more potent than VEGF and has fewer side-effects, particularly the risk of oedema. In Phase 1 and 2 trials, intramuscular administration of HGF in patients with CLI improved ulcer healing, rest pain and ABPI.27 These beneficial effects were maintained even after a two-year follow-up, encouraging further clinical trials. Hypoxia-inducible factor 1- alpha (HIF 1α)28 and developmental endothelial locus-1 (DEL-1)29 are the other two angiogenic factors which showed promising results in pre-clinical studies but failed to show clinical benefit in Phase 1 and 2 clinical trials.
The other well-explored option in therapeutic angiogenesis is stem cell therapy. The key concept is that stem cells can proliferate and generate new blood vessels in ischaemic tissues. Stem cells can be subclassified by their lineage: embryonic stem cells (ESC) and adult stem cells (ASC). Of particular interest in the context of angiogenesis is the endothelial progenitor cell;30 however, this term has fallen out of favour and instead it is now more common to refer to mononuclear cells (MNC), either harvested from bone marrow (BMMNC) or peripheral blood (PBMNC).
Two landmark trials reported the use of stem cells: TACT and PROVASA. TACT31 showed that injection of BMMNC into the gastrocnemius muscle led to an improvement in CLI. In its second stage, TACT also investigated whether effects brought about by BMMNC were superior to those from PBMNC. At 4 and 24 weeks, there were significant improvements in ABPI, rest pain and walking time. PROVASA,32 on the other hand, demonstrated that intra-arterial administration of BMMNC did not lead to significant increases in ABPI. Nonetheless, there were notable improvements in ulcer healing and rest pain at 6 months. More recently, Inei and colleagues have reported 4-year data on amputation-free survival in patients with CLI or Buerger's disease after intramuscular injection of BMMNC. Of note, amputation rates were halved in the CLI group (100% vs 52%). Most recently, a meta-analysis of 12 randomised trials33 has shown improvements in amputation (8 treatments to prevent a single event), ABPI, ulcer healing and walking distance, following stem cell therapy. For these reasons, stem cell therapy is the most promising new technology in the management of peripheral arterial disease.
5One of the limitations of BMMNC therapy is the need for invasive and painful bone marrow aspiration to procure the cells. PBMNC therapy aims to overcome this limitation by mobilising BMMNC to the peripheral blood using granulocyte-colony stimulating factor (G-CSF) and harvesting them from the peripheral blood circulation. The therapeutic potential of these peripherally-harvested MNC were evaluated in few clinical trials. Huang et al 34 and Ozturk et al 35 performed autologous transplantation of PBMNC in diabetic patients with CLI and found that the pain scores and ABPIs were significantly improved. In another small clinical trial, Mohammadzadeh et al reported improved ABPIs and amputation rates by transplanting autologous PBMNC in diabetic patients with CLI.36 A direct comparison of the therapeutic potential of BMMNC and PBMNC was performed in a few clinical trials with variable results.3739
 
CHRONIC FOOT ULCERS
In the studies described in the preceding section, it is clear that stem cells have shown great translational potential in the management of peripheral arterial disease. There is some evidence that they may also have a future in ulcer healing, beyond improving limb vascularity. However, this area of research is not as advanced as that of therapeutic angiogenesis and much of the evidence for the use of stem cells to treat lower limb ulcers comes from preclinical models and small clinical case series.
The role of stem cells in foot ulcers is not only to propagate and differentiate into skin tissues but also to promote an environment where the native cells may proliferate and eventually cover the ulcer area. Analysis of animal models of stem cell transplantation has shown that this is achieved by the release of a cascade of growth factors including vascular endothelial growth factor and epithelial growth factor.40 The choice of stem cell is just as important in ulcer management as it is in angiogenesis. Embryonic stem cells are a default option given their pluripotent ability to differentiate into any cell type. However, this treatment raises a number of ethical controversies, given the need to sacrifice human embryos to procure the cells. Hence, an obvious alternative is autologous bone marrow as a source of stem cells.
Kataoka41 conducted an elegant experiment where bone marrow mesenchymal stem cells were harvested from green fluorescent protein (GFP) transgenic mice (a breed of mice that have been genetically engineered so that their cells express proteins which fluoresce under the appropriate light wavelengths) and transplanted onto wounds on genetically bald mice. After three weeks, the wounds had healed and hair was growing in the transplantation area. Furthermore, histological analysis of the treated skin revealed GFP visible in the dermis, epidermis and hair follicles, demonstrating that the stem cells had successfully differentiated into multiple cell lines. The effectiveness of bone-marrow derived mesenchymal stem cells has been shown in other murine models42 and in a small series of 8 patients,43 which demonstrated that bone-marrow derived mesenchymal stem cells helped to reduce ulcer size. The use of bone-marrow derived haematopoietic stem cells also led to positive effects on ulcer healing, when applied topically,44 in a murine model.
6There has also been some interest in the use of adipose-derived stem cells (ADSC). These are harvested by performing liposuction and using selection enzymes to separate them from ‘contaminating’ adipocytes.45 Studies with ADSC have not reached clinical trials, though early animal data have shown improvements in wound healing in different preclinical models.46
The above studies have investigated the topical application of stem cells to wounds. However, the simple application of stem cells to a wound bed is insufficient to promote healing; the ulcer environment is, by default, hostile to the cell through a combination of inflammation, ischaemia and oxidative stress. Biological scaffolds are being increasingly tested to encourage both cell seeding and their uptake on the ulcer bed. Fibrin scaffolds are marketed as haemostatic sealants,47 and as such are perhaps the most widely available. However, there is evidence to suggest that these inhibit the proliferation of stem cells, and their use has not led to uniformly positive results.48 Another approach is to use collagen scaffolds. Collagen is one of the most abundant components of the extracellular matrix in mammals. There are 16 types of collagen; skin and vascular tissues are predominantly composed of types I and III. Collagen is laid down in staggered, end-to-end arrangements, as well as by cross-linking, giving collagen-containing tissues a high tensile strength. Hence, applying the stem cell within a collagen-based gel is analogous to transplanting a cell within its native biological scaffold. Indeed, collagen biogels have been shown to bring about enhanced engraftment of stem cells in animal models.49 However, this has not yet been translated into human studies.
An alternative approach is to manage wounds primarily with extracellular matrices without the contemporaneous use of stem cells. Such matrices can be biological scaffolds as described earlier, or decellularised matrices (DCMs). The mechanism of action of DCMs is not fully understood but it has been suggested that they work by binding matrix metalloproteinases (enzymes which work to breakdown the wound bed) and nullifying their biological effects. It is also thought that DCMs promote the recruitment of epithelial cells and vascular endothelial cells. Sources for DCMs include porcine intestinal submucosa and human dermal matrices, many of which are available commercially. Whilst no randomised controlled trials have been carried out to assess the effectiveness of these products, the manufacturer-sponsored literature demonstrates encouraging results. However, it is difficult to judge these studies without proper controls; in a study of 23 patients who were treated with surgical wound debridement and topical application of equine pericardium, there was a 94% wound improvement rate.50 However, it is not clear to what extent this was brought about by debridement alone.
The true clinical effect of DCMs remains elusive. One of the most recent advances in ECM engineering has been the development of hybrid biological/synthetic matrices such as the Integra Dermal Regeneration Template.™ This template combines a glycogen and glycosaminoglycan (dermal) layer with a silicone (epidermal) layer. These templates have been predominantly developed for the management of burns,51 but recent evidence suggests that they may also have an application in the management of the diabetic foot ulcer.52
7The gold standard in the management of foot ulcers will continue to be infection control, offloading and revascularisation of ischaemic tissues. However, there are clearly emerging technologies and treatments on the horizon which may help to manage more effectively this frequent and disabling complication of peripheral vascular disease.
 
ANEURYSMAL DISEASE
The surgical management of aortic aneurysmal disease has fundamentally changed over the last two decades. Lessons learnt from the UK small aneurysm study,53 MASS trial54 and EVAR 1 and 2 trials55 have served to reduce the mortality associated with abdominal aortic aneurysm rupture and interventions carried out to prevent it. Nonetheless, the fundamental biology of aneurysm development (as opposed to its risk factors), still eludes most clinicians.
The aorta contains a high proportion of collagen and elastin, which serve to give it tensile strength and elasticity, both of which are vital for the carriage of pulsatile blood. Histological studies have demonstrated that aortic elastin is altered in Marfan's syndrome56 and in the elderly population.57 Additionally, there is a lower concentration of elastin in aneurysmal aortas compared to healthy vessels.58 It is, therefore, natural to presume that there is an association, if not causation, between collagen, elastin and aneurysm development.
The variability in collagen types (as described in the ulcer management section) has led to studies of the differential composition of collagen in aneurysmal and healthy aortas. In particular, there has been an interest in the two most common types: I and III. Bode59 has described the distribution of collagen types in aneurysmal aortas: his group found that the media from abdominal aortic aneurysms had a higher concentration of type III collagen than those from healthy aortas. Similarly, there is some evidence of a higher rate of collagen III breakdown in patients with aneurysmal disease, as evidenced by higher circulating concentrations of its fragments.60,61
The changes in collagen and elastin composition in aneurysmal aortas have driven researchers to investigate the mechanism behind their catabolism in the hope of inhibiting this process. Matrix metalloproteinases (MMPs) are a group of zinc-dependent enzymes responsible for the degradation of extracellular proteins. Interstitial collagen degradation is associated with increased expression of the collagenases MMP 1 and 13, whilst the elastases MMP 2, 9 and 12 have been noted in the wall of aneurysmal aortas. As MMPs have also been linked with tumour invasion, the study of MMP inhibition is well established in the field of cancer research. MMP inhibitors work by targeting the catalytic zinc ion within the enzyme with a number of zinc-binding molecules demonstrating in vitro and in vivo MMP inhibition.62 However, to date, Phase 3 trials have mostly met with disappointing results,63 often due to the side-effect profiles of these compounds.64
In recent years, aneurysm researchers have become interested in the MMP inhibitory effects of tetracyclines, a discovery that was made and developed in the field of periodontal disease.65 Petrinec66 was among the first to study the effects of doxycycline on mice. He demonstrated a 8significant reduction in the rate of aneurysm development and growth in an experimental model. Further work in this field successfully explored the use of low-dose regimes67 before translating into human studies.68 However, trials exploring this therapeutic strategy have not shown unanimously positive results.69 Initial results were encouraging, with significant reductions in aneurysm expansion, but the Pharmaceutical Aneurysm Stabilization Trial revealed no difference in aneurysm expansion or rate of repair over 18 months in patients taking doxycycline.70 Nonetheless, the effectiveness of doxycycline in modulating aneurysm development is still of interest to the clinical community with a Phase 2 North American randomised trial currently recruiting patients.
Another strategy for the attenuation of aneurysms is the inhibition of elastases. As has been established, elastin content is altered in aneurysmal aortas. Indeed, pancreatic elastase infusion is an established method of developing aneurysms in animal models.71 Elastase inhibition has historically been of interest for the prevention of emphysema72 and in wound healing.73 However, it is only recently that the effects of elastin inhibitors have been studied for aneurysmal disease. Delbosc and colleagues have published an interesting animal study using a rat model of aneurysm development, wherein the administration of a synthetic elastase inhibitor led to an attenuation in infective (Porphyromonas gingivalis) aneurysm progression.74 They propose that it may have a role in the future management of aneurysms, though it is worth noting that such inhibition was not observed in the absence of infection, and the management of mycotic aneurysms presents an entirely different set of clinical challenges.
In summary, the pathophysiological processes responsible for the development of aneurysms are not as well understood as those of atherosclerotic disease. However, there appears to be a clear role for the catabolism of collagen and elastin. The enzymes that degrade these proteins have been implicated in the development of aneurysmal disease, but the inhibition of these enzymes has not yet led to clinically significant improvements in outcomes. As the evidence to date is equivocal, the results of large clinical trials are awaited. Future research could elucidate the missing links in the development and medical management of aortic aneurysms.
 
CONCLUSION
This chapter has described some of the important advances in research in the management of diseases commonly encountered by vascular surgeons. The overview of the four research topics covered is by no means comprehensive; rather, it is designed to provide readers with an introduction to the areas of ongoing vascular research. Most of the therapies discussed are some years away from entering routine clinical practice. Clinical efficacy and cost will be important factors in bringing these therapies to market. However, ‘a journey of a thousand miles begins with a single step’: many modern clinical therapies would not have been possible without translational research.
9Clinical and pre-clinical research depends crucially on external funding and the provision of such financial support remains a challenge, with a multitude of scientists and clinicians competing for funds from what seems to be an ever-decreasing pool of resources. Even the concepts which show great initial promise may not progress to the bedside, i.e. move ‘from concept to clinic’, without adequate and timely funding. On a positive note, we are witnessing greater collaboration between surgeons and scientists with each contributing to, and enhancing, the other's work. Such collaboration is at the heart of translational vascular research.
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