Abstract
Advances in mouse and human genome research have dramatically altered the landscape and pace of advances in the diagnosis and treatment and many diseases. Peripheral arterial disease (PAD) is a major complication of systemic atherosclerosis and healthcare problem. Genetic and genomic research has done little to alter the diagnosis and management PAD, but this likely will change in the near future. This chapter will examine how rodent and human genetics in PAD may soon guide advances and eventually personalized therapies.
Keywords
Peripheral arterial disease, therapeutic angiogenesis, gene therapy, modifier gene, gene polymorphism, single nucleotide polymorphism, linkage disequilibrium, haplotype, gene association studies, genome wide association studies, quantitative trait locus, genomic methodology
Chapter Outline
Introduction 197
Epidemiology and Risk Factors for PAD 198
Clinical Manifestations of PAD: A Barrier to PAD Genetics 200
Therapeutic Strategies for PAD 201
Genetic Susceptibility in the Causation of PAD 202
Genetic Modifiers of PAD 207
Refining an Identified QTL Using Haplotype Analysis 208
Identification of Candidate Genes using Expression Data 209
Biomarkers of PAD 210
Future Potential Use of Genomic Methodologies in PAD 211
References
Further Reading 219
Introduction
Information from rodent and human genome projects have resulted in logarithmic advances in mapping and identifying new disease-related genes. Methodologic advances have increased the speed and accuracy and lower the cost of genome research which has enhanced the opportunity to develop novel diagnostic and therapeutic approaches across a host of human diseases. Not only through the identification of novel disease-related genes but also to understanding the effect of gene modulation in disease processes. The ultimate goal is the identification of markers that facilitate gene detection or allow for the guidance and targeted strategies for therapeutic interventions. Such is a goal of personalized medicine.
Genetic advances and clinical application has not been uniform across all disciplines in medicine. Cancer research has been forever changed by genetic advances but not so in all fields. In vascular biology, and more specifically in peripheral arterial disease (PAD), investigations are beginning to employ genetic approaches but the opportunities far exceed the amount of knowledge secured to date. Here we will describe the epidemiology, risk factors, and clinical presentations of PAD. We will report on some of the future avenues through which genomic methodologies might be used to further our understanding of PAD; a disease that remains vastly understudied despite its prevalence and severity of clinical manifestations.
Epidemiology and Risk Factors for PAD
PAD is defined as an obstruction in a major arterial bed other than the coronary arteries and PAD is caused by atherosclerosis in well over 90% of patients . The most common site for PAD is the lower extremity and commonly PAD is equated to the condition of PAD of the lower extremity, where occlusive atherosclerosis leads to impaired perfusion to the leg or legs. The risk factors for the development of PAD in many ways can be considered as the same as those recognized as important in any form of systemic atherosclerosis, and include increasing age, cigarette smoking, diabetes mellitus, hyperlipidemia, and hypertension, in decreasing order of magnitude ( Table 12.1 ) . Elevated levels of C-reactive protein (CRP) and homocysteine may also be important risk factors . Despite the similarity in risk factors to coronary artery disease (CAD), smoking and diabetes impart the vast majority of the age-adjusted increase in risk, and an age >40 is generally required to make the diagnosis .
| Intermittent Claudication (IC) | Critical Limb Ischemia (CLI) | |
|---|---|---|
| Risk Factors | ||
| Diabetes | Known risk factor | Known risk factor |
| Smoking | Known risk factor | Known risk factor |
| Hypertension | Known risk factor | Known risk factor |
| Hyperlipidemia | Known risk factor | Known risk factor |
| C-reactive protein | Known risk factor | Known risk factor |
| Clinical Characteristics | ||
| Pain with ambulation | By definition pain, ache, and heaviness | Variably resent |
| Pain at rest | Not present | Usually present |
| Ulceration or gangrene | Usually not present | May be present |
| ABI <0.9 | Usually present | Sometimes associated with lower ABIs, but low ABI does not predict disease |
| Annual mortality | 1%–2% | 20% |
| Annual amputation rate | <1% | 25%–40% |
| Biochemical Characteristics | ||
| B2 Microglobulin | Higher levels in PAD patients, but not shown to differentiate between IC and CLI a | |
| Angiogenic Factors or Receptors | Higher levels of sTie 2 in PAD patients, and also shown to differentiate between IC and CLI b | |
a Wilson, A. M., Kimura, E., Harada, R. K., Nair, N., Narasimhan, B., Meng, X. Y., Zhang, F., Beck, K. R., Olin, J. W., Fung, E. T. & Cooke, J. P. (2007) Beta2-microglobulin as a biomarker in peripheral arterial disease: proteomic profiling and clinical studies. Circulation , 116, 1396–403.
b Findley, C. M., Mitchell, R. G., Duscha, B. D., Annex, B. H. & Kontos, C. D. (2008) Plasma levels of soluble Tie2 and vascular endothelial growth factor distinguish critical limb ischemia from intermittent claudication in patients with peripheral arterial disease. J Am Coll Cardiol , 52, 387–93.
PAD has been underrecognized and underdiagnosed by the medical community and, either by cause or effect, viewed as less important than heart disease. Decade old data showed that PAD had a population prevalence that approached 3/4 that of ischemic heart disease and recent data has confirmed this finding. PAD affects 3%–10% of adults in the world and 15%–20% of those over 70 years. With an aging population one would expect that the prevalence of PAD will continue to increase .
Clinical Manifestations of PAD: A Barrier to PAD Genetics
The principle problem in PAD is leg perfusion that is insufficient to meet tissue demands either at rest or with exercise. There are two major clinical manifestations of PAD: intermittent claudication (IC) and critical limb ischemia (CLI) (see Table 12.1 for comparison of IC and CLI). In IC reduced blood flow to the leg(s) with exercise results in calf or thigh pain or aching relieved by rest. In CLI patients, pain at rest that may be associated with nonhealing leg ulcers or gangrene, though patients can have ischemic ulcers without leg pain. Patients with IC have amputation and annual mortality rate of 1%–2%, those with CLI have a 6-month amputation risk of 25%–40% and an annual mortality of 20%, and the conversation rate from IC to CLI is insufficient to account for the number of patients with CLI . This is the first area where genetics may play a role because an occlusion can cause an obstruction but a gene(s) may modify the clinical outcome once the occlusion is present ( Fig. 12.1 ).
The lack of expected clinical progression for PAD patients going from IC to CLI limits the ability to use retrospective population data sets to look for markers. In addition retrospective data sets may lack the diagnostic accuracy for PAD because even when prospectively applied, most patients who meet the definition for having PAD, either lack the classic symptoms of PAD or lack any symptoms at all. Some estimates suggest that as many as 50% of PAD patients fall into this category . These patients can be labeled as asymptomatic from a clinical perspective but the absence of classic PAD symptoms cannot be equated with the absence of disease. For population studies, this is especially problematic true when one considers that PAD occurs a decade or more later than CAD.
The diagnosis of IC versus CLI is based upon time-tested clinical classification schemes, namely the Rutherford and the Fontaine classifications. In the Rutherford classification, IC encompasses categories 1–3 (mild, moderate, and severe claudication, respectively), while CLI includes categories 4–6 (ischemic rest pain, minor tissue loss, and ulceration or gangrene). The Fontaine classification is more commonly used in Europe, with stages IIa and IIb describing IC, while stages III–IV are categories of CLI.
As for any disease the “screening” of potential patients, in the absence of obvious symptoms, will influence disease prevalence. The simple, noninvasive ankle brachial index (ABI) is used to diagnose PAD . The ABI uses simple hand-held Dopplers to measure the ratio of systolic blood pressure in the ankle to that of the brachial vessels. An ABI <0.9 is considered diagnostic for PAD . Once PAD is diagnosed, further testing with duplex ultrasonography, segmental Doppler pressure or volume plethysmography, MRA, or angiography may be utilized, depending on the clinical situation. There is a lack of uniformity on the role of screening even high-risk patients (smokers and diabetics) as there is a lack of clinical benefit in the treatment of PAD.
Therapeutic Strategies for PAD
Current treatments for peripheral artery disease (PAD) can be considered as those which target the leg and those seek to reduce complications of stroke and myocardial infarction (MI) from systemic atherosclerosis. Systemic targeted therapies include those for the treatment of hypertension, dyslipidemia, as well as the use of antiplatelet therapy. A recent paper though did question the use of aspirin in patients with PAD; though at the time of writing this chapter, aspirin (ASA) currently has a Class 1A indication using American Heart Association/American College of Cardiology guidelines .
Medical therapies aimed at reducing leg symptoms, i.e., increasing walking time in patients with established claudication include use of the phosphodiesterase inhibitor cilostazol . The methylxanthine derivative, pentoxyphilline, improves deformity of red blood cells, is also used, but has been shown to be less effective . There are no medical therapies for CLI . There are no pharmacological therapies available for PAD that have the ability to increase perfusion to the leg and thus correct the principal underlying problem of reduced blood flow.
Still today, exercise training is the most effective PAD treatment for improving symptoms and increasing walk time in IC . Supervised exercise is now recommended as a key component of PAD management . The molecular mechanism by which exercise improves symptoms and walking time in PAD is likely multifactorial but includes the growth of blood vessels, i.e., angiogenesis. Simply bypassing a blockage should be an easy and effective treatment strategy for any PAD patient and surgical and catheter-based revascularization strategies, or a combination of both, are dramatically increasing in frequency, especially in the United States . Interestingly, problems remain in this simple approach. First, not all patients are candidates for revascularization, due to a variety of reasons, including small target vessels, diffuse PAD, and presence of other comorbidities. Second, procedural failure rates remain high and especially in the patients with distal disease who are the ones most in need of treatment. Third, in patients with IC, exercise is at least as good if not better than revascularization . The lack of conventional medical therapies combined with the limited success of revascularization has led to the investigation of novel therapeutics.
Therapeutic angiogenesis is an investigational approach in which growth factors are delivered to ischemic tissues in an attempt to improve perfusion. A variety of cytokine growth factors are currently being, or have been, investigated. The vectors used to facilitate the therapy have varied from simple to modified protein, plasmid DNA, or viral vectors. Adeno-associated viruses have led to major advances in some human diseases and may eventually be useful in patients with PAD .
Therapeutic angiogenesis studies in PAD have used primarily vascular endothelial growth factor (VEGF), fibroblast-derived growth factor (FGF), hepatocyte growth factor (HGF), and angiopoietins . A Phase I/II clinical study of HGF-plasmid in CLI patients unsuitable for revascularization showed a significant increase from baseline in transcutaneous partial pressure of oxygen (used as a measure of limb perfusion) in the high-dose group compared with placebo at 6 months. Larger trials will be needed to determine whether HGF-plasmid can improve wound healing, limb salvage, or survival . NV1FGF is a modified plasmid DNA delivery system that sought more efficient and sustained local expression of FGF-1. Previously, the double-blind, randomized, and placebo-controlled Phase II TALISMAN study, conducted in Europe, demonstrated a significant reduction in the risk of major amputation for CLI patients treated with IM NV1FGF . However, data presented at the Phase III TAMARIS study, which was designed to evaluate the safety and efficacy of NV1FGF in 525 CLI patients with skin lesions (due to ulcer or gangrene) who were unsuitable for revascularization (ClinicalTrials.gov Identifier: NCT00566657) was disappointing. At 1 year, no significant difference was observed between the NV1GF group versus the placebo arm in the primary end point of time to major amputation or death .
Genetic Susceptibility in the Causation of PAD
Beyond smoking and diabetes, is there evidence that an individual’s genetic background may be important in causing PAD. In one study, the prevalence of PAD in selected ethnic backgrounds suggested higher prevalence in African–Americans (AAs), even after adjusting for age and other traditional PAD risk factors . This suggests that gene polymorphisms may contribute to the development of PAD.
Data is available from association and linkage studies where estimates of the heritability of PAD, using ABI, have shown that the contribution of genetic factors to overall variation in ABI ranges from 21% to 48% . In the Multi-Ethnic Study of Atherosclerosis, self-reported race/ethic group predicted the presence of PAD, independent of all other “established” and “novel” cardiovascular disease risk factors of atherosclerosis, with the lowest risk in Hispanics and Chinese and the highest in AAs .
Gene Polymorphisms and Development of PAD
Genes that cause PAD can be classified into three different categories: proatherosclerotic, proatherothrombotic, or unknown, based on the knowledge (or lack thereof) of the function of the gene products ( Table 12.2 ). Since PAD is a consequence of atherosclerosis in the lower extremities, any genetic polymorphisms that increases the risk of developing atherosclerosis could/should be associated with PAD. In regards to hyperlipidemia, Monsalve and coworkers evaluated polymorphisms within the APO B gene among 205 patients with a diagnosis of PAD. Their data showed higher prevalence of an R2 and X1 allele among the PAD patients compared to non-PAD controls. They concluded that variations at the apo B locus contributed to predisposing individuals to the development of arterial disease, but they could not specify PAD . An association study from the Honolulu-Asia Aging Study involved 3161 Japanese–American men aged 71–93 showed association of APO E polymorphism with PAD . Any mechanism by which a genetic polymorphism contributes to the pathogenesis of PAD is poorly understood.
| Thrombosis | Atherosclerosis | Unknown |
|---|---|---|
| Factor II (FII G20210A) | APO E | PAOD1 |
| P2Y12 (H2 allele) | APO B | |
| Fibrinogen (beta) | IL-6 promoter (−174 G/C) | |
| E-Selectin Ser128Arg | ||
| ICAM-1 (469E/K) | ||
| MCP-1 (−2518 A/G) | ||
| MMP1 and MMP3 | ||
| eNOS (−786C) | ||
| ACE D | ||
| CHRNA3 |
Inflammation is becoming increasingly recognized as an important factor in the pathogenesis of the development and progression of atherosclerosis being orchestrated by several cytokines, chemokines, adhesion molecules, and proteolytic enzymes . IL-6 is a proinflammatory, multifunctional cytokine produced by various cell types, including monocytes, adipocytes, and endothelial cells. Elevated levels of IL-6 have been found in patients with atherosclerotic disease . Sequence variations in the promoter region of the IL-6 gene (−174) have been reported with two different alleles identified resulting in three genotypes: GG, GC, and CC that influence IL6 transcription and plasma levels . Flex et al., in their study of 84 patients with PAD, found that the GG genotype was more common in individuals with PAD, suggesting a role for this proinflammatory cytokine in the pathogenesis of PAD .
In a study of 157 PAD patients and 206 controls, Flex et al. analyzed gene polymorphisms including IL-6 ( − 174 G/C ), E-selectin ( E-sel ) Ser128Arg , intercellular adhesion molecule-1 ( 469 E/K ), monocyte chemoattractant protein ( MCP-1 )— 2518 A/G , matrix metalloproteinase ( MMP)-1—1607 1G/2G , and MMP-3—1171 5A/6A . The IL-6 , MCP-1 , and MMP-3 polymorphisms influence the rate of transcription of the respective genes, substantially influencing protein plasma concentrations. MMP-1 plasma levels are also significantly affected by the polymorphism studied. The ICAM-1 polymorphism changes the amino acid sequence of the immunoglobulin-like domain 5, which is important for intracellular adhesion molecule (ICAM)-1 and leukocyte antigen interactions, and for B-cell adhesion. They found that IL-6 , E-sel , ICAM-1 , MCP-1 , MMP-1 , and MMP-3 gene polymorphisms were significantly and independently associated with PAD. They also found that these proinflammatory polymorphisms act synergistically and determine genetic profiles that confer different levels of risk for PAD and CLI, depending on the number of high-risk genotypes concomitantly present in a given individual. This is an instance of how susceptibility to a disease results from functional interactions among a number of modifier genes .
Angiotensin-converting enzyme (ACE) generates angiotensin II, a potent hypertensive factor. The ACE insertion/deletion (I/D) polymorphism refers to a 287-bp fragment in intron 16 of the ACE gene. The ACE D allele is associated with increased serum levels of the circulating enzyme . Basar et al. and Li et al. found borderline significant association between ACE I/D polymorphism and PAD . Fatini et al. studied the ACE I/D polymorphism and the − 240 A>T polymorphism in the promoter region of the ACE gene in the context of PAD, and showed that the ACE D allele and the ACED/ − 240T haplotype significantly and independently influenced predisposition to PAD .
ACE may also affect bradykinin degradation and nitric oxide (NO) release . NO is synthesized from l -arginine by three isoforms of NO synthase (NOS), with endothelial being most relevant to vascular disease (eNOS) . eNOS-derived NO acts as an antiatherogenic molecule and prevents the progression of atherosclerosis . eNOS gene polymorphisms have been shown to cause decreased NO synthesis, thereby reducing NO availability and causing endothelial dysfunction . Of the several eNOS gene polymorphisms, the − 786T>C and eNOS 4a4b polymorphisms are associated with variability in the NO plasma levels . The − 786C allele significantly reduces eNOS promoter activity, and individuals carrying the 4a4a genotype were found to have lower NO production . Fowkes et al. did not find an association between eNOS 4a/4b polymorphism and PAD . However, Sticchi et al. demonstrated an association between eNOS and ACE genes in increasing the susceptibility to PAD in smokers, providing an evidence for a modifier gene–environment interaction. In smokers, but not in nonsmokers, the concomitant presence of the eNOS − 786C and 4a alleles was significantly associated with increased predisposition to PAD. Furthermore, the eNOS-786/4a haplotype also increased susceptibility to PAD in smokers (but not in nonsmokers) also carrying the ACE D allele .
Polymorphism of Proatherothrombotic Genes and PAD
Thrombosis plays an important role in the pathogenesis of atherosclerosis . Thrombin is important in fibrin formation, platelet aggregation endothelial activation, and leukocyte recruitment . Studies looking at fibrinogen gene polymorphism found that certain genotypes were associated with an increased risk of peripheral atherosclerosis . Reny et al. investigated an association of factor II and V polymorphisms in PAD . Although no association was found for factor V polymorphisms, a statistically significant association was shown between the FII G20210A allele of factor II which is linked to increased local thrombin generation and PAD. The platelet ADP receptor P2Y12 is a 7-transmembrane receptor which upon activation promotes platelet aggregation . Polymorphism in the P2Y12 gene has been described ; one of the alleles ( H2 ) results in a gain of function haplotype on ADP-induced platelet aggregation. Hence, variations in this allele may be associated with increased risk of PAD even after adjustment for traditional PAD risk factors .
Thrombophilia can also be mediated through defects in the folate pathway . Methylenetetrahydrofolate reductase is a critical enzyme in the folate/homocysteine pathway. Three studies found a significant positive association between MTHFR 677C/T polymorphism and PAD .
Recent studies have shown that loss of function mutation in the gene encoding the facilitative glucose transporter GLUT10 ( SLC2A10 ) causes arterial tortuosity syndrome via upregulation of the transforming growth factor-β. The SLC2A10 gene is thought to be a candidate gene for vascular complications in type 2 diabetics . In a prospective cohort study of 372 diabetic patients, SNPs of the SLC2A10 gene were significantly associated with PAD, with the strongest association shown by the T allele at rs2179357 . They also identified a common haplotype (H4) conferring a strong risk of PAD in type 2 diabetic patients and carriers of the H4 haplotype were more likely to develop PAD during follow-up .
Unbiased Genetic Locus and PAD Susceptibility
Human studies of the genetics of PAD are actually quite limited, outside of genes contributing to atherosclerosis or thrombosis. Only a single family-based linkage study that has identified a genetic locus conferring susceptibility to PAD exists. This study of Icelandic families with multiple family members exhibiting PAD identified a locus termed peripheral arterial occlusive disease ( PAOD1 ) that mapped to human chromosome 1p31 . Interestingly, other risk factors for PAD, such as hypertension, hyperlipidemia, and diabetes, did not contribute to the positive linkage. The gene(s) responsible for PAOD1 have not been identified. A few genome-wide association studies (GWAS) and PAD have been published ( Table 12.3 ). Smoking is a critical risk factor for PAD and one study found a significant association between nicotine dependence and a SNP ( rs1051730 ) on chromosome 15q2. This SNP is located within the CHRNA3 gene, which is in a linkage disequilibrium block that contains genes encoding nicotinic acetylcholine receptors. Interestingly, this study also demonstrated a significant association of this SNP with PAD and lung cancer, suggesting a gene–environment interaction .
