In homeostasis, leukocytes move in and out of the vascular system and tissues and are in constant surveillance of their microenvironment waiting for a signal. In response to stimuli, these cells are recruited to inflamed tissues, where they “clean up” the injury and contribute to repair. In atherosclerosis, damage to the endothelium (e.g., increased turbulence of blood flow, high blood pressure, high cholesterol, high glucose, oxidation etc.,) can upregulate multiple mediators governing leukocyte recruitment. Chemokines, cytokines, and other inflammatory mediators regulate the expression of adhesion molecules on both the endothelium, neutrophil, and monocyte surface to influence the three-step process of leukocyte recruitment: rolling, activation, and adhesion on the endothelium which involves E-, L- and P-selectins. While rolling is essential for leukocyte adherence, it does not necessarily lead to firm adhesion; firm adhesion requires activation of integrins (by selectin or chemokine engagement) and their interaction with ICAM-1 and VCAM-1 (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1), which results in the complete arrest of the leukocyte. Leukocytes then transmigrate between the endothelium into the interstitial space toward a chemotactic stimulus, such as, CC-chemokine ligand-2 (CCL-2) (14).

Some of these cellular interactions have been implicated in PAD. For example, circulating levels of monocytes are significantly and independently associated with PAD (15), with high neutrophil-lymphocyte ratio a potential predictor of PAD severity (16, 17). Circulating chemokines and inflammatory markers expressed by leukocytes including high sensitivity C-reactive protein (hs-CRP), interleukins (IL), and matrix metalloproteinases (MMPs) are also upregulated in PAD patients, and in some cases associate with severity of disease (18–21). Indeed, a systematic review and meta-analysis of 47 studies involving >21,000 PAD patients, identified high levels of hs-CRP to predict the risk of major adverse cardiovascular events and mortality (22). Platelets release low abundance, highly active molecules including chemokines/chemokine ligands and angiogenic factors including chemokine ligand-5 (CCL-5) and platelet-derived growth factor (PDGF), which support leukocyte-platelet interaction and migration of neutrophils and monocytes to the developing atherosclerotic site (23). Indeed, Barrett et al., recently identified that platelets induced the migration of monocytes into atherosclerotic lesions of Ldlr^−/− mice via upregulation of monocyte suppressor of cytokine signaling 3 (SOCS3) (24). Thus, platelets also contribute to inflammation, and with increased inflammatory burden, there is an increase in PAD prevalence (25). Another cytokine important for migration of leukocytes into the vessel wall is CCL-2. CCL-2 infusion of the femoral artery of rabbits following hindlimb ischemia increased monocyte accumulation in the vessel wall (26); a finding that was inhibited with ICAM-1 monoclonal antibody treatment (27), suggesting that monocyte adhesion to the endothelium in ischemia involves CCL-2 and ICAM-1. Importantly, circulating CCL-2 levels are increased in PAD (18, 28), associating with increased CCL-2 protein expression and macrophage content in limb tissues from patients (29).

Additional reports of the role of cell adhesion molecules come from the Edinburgh Artery Study, which described increased soluble levels of ICAM-1 associating with PAD diagnosis (30). In another study, soluble VCAM-1 levels were associated with worse PAD prognosis (31). From the selectins: E-selectin is EC specific whereas P-selectin is expressed by both ECs and platelets. Higher levels of soluble E-selectin have been reported in PAD patients, particularly in diabetes, and reflecting endothelial activation (32). Circulating P-selectin levels are also associated with PAD severity (33–35) and in the Multi-Ethnic Study of Atherosclerosis, a prospective large cohort study involving >6,800 participants, P-selectin levels were significantly associated with lower ankle-brachial index ratios as well as PAD prevalence (36). P-selectin's involvement in leukocyte adhesion was confirmed in vitro; human recombinant P-selectin increased neutrophil adhesion to platelets in the presence of plasma from healthy individuals (34), suggesting that neutrophil adhesion could also occur with activated endothelium expressing P-selectin in PAD patients. Moreover, a link between platelets and SOCS3-mediated activation in PAD was observed, with the SOCS1:SOCS3 ratio negatively correlating with IL-1β, but also with monocyte-platelet aggregates, P-selectin and CD40 (24). Indeed, increased circulating leukocyte-platelet aggregates are proposed as a biomarker of PAD severity (17). An in-depth summary of inflammatory biomarkers in PAD was recently reviewed (37). Figure 1 summarizes the endothelial, leukocyte and platelet contribution to inflammation in PAD.

Under normal circumstances, the endothelium exquisitely controls endothelial-platelet interactions and the balance between coagulation and anticoagulation in the vessel wall. ECs generate nitric oxide (NO) and prostacyclin, molecules which directly inhibit platelet activation. They express tissue factor pathway inhibitor, a potent anticoagulant, which limits tissue factor-inducible activation of factor VII and X (38). ECs also express co-factors for antithrombin III, or synthesize thrombomodulin (a thrombin receptor), which can directly reduce plasma thrombin levels upon thrombin binding, an effect that can increase the activity of the anticoagulant, protein C (39). Furthermore, ECs secrete tissue-type and urokinase-type plasminogen activator (t-PA and u-PA, respectively) to activate fibrinolysis and fibrin degradation. Because thrombosis is strongly implicated in PAD (6), the EC-platelet interaction may be more central to PAD pathogenesis. Indeed, PAD patients have elevated levels of circulating platelets (40) and increased platelet aggregability (41), with mean platelet volume increasing with PAD severity (42). Circulating tissue factor (43), t-PA (40) and factors IX and XI (44) are also increased, whereas tissue factor pathway inhibitor (43) and protein C levels are decreased (44); the latter associating with endothelial injury (44). Elevated von Willebrand factor and fibrinogen levels are also independently associated with the risk of development of PAD (45), with increased fibrinogen and D-dimer levels predictive of increased risk of mortality in PAD patients (22). Soluble thrombomodulin levels are increased in symptomatic PAD vs. asymptomatic age-matched control subjects (46), which is significant since elevated levels may reflect EC dysfunction in PAD (47, 48).

Interestingly, medial calcification and calcified nodules were identified in 70% of CLTI peripheral arteries examined (6, 49). Calcified nodules, accompanied by fibrin could pierce or disrupt the fibrous cap causing EC loss and plaque rupture (50). This is important since vascular calcification may predict poorer outcomes in PAD (51). However, reports suggest that calcification may also stabilize plaque (52). The role of calcification in PAD is not well established and requires further study.

To meet physiological demand and manage blood flow, the endothelium dilates arteries by relaxing the underlying smooth muscle via a range of mechanisms. The most well studied is NO. Under normal physiological conditions, the formation of NO is dependent on calcium facilitated calmodulin binding to homodimeric endothelial nitric oxide synthase (eNOS). Bound calmodulin then facilitates, 6R-tetrahydrobiopterin (BH4)-dependent electron transfer across eNOS to catalyze the conversion of L-arginine to NO and L-citrulline. However, roles for cyclooxygenase (COX)-derived prostoglandins (53), membrane hyperpolarization (54), epoxyeicosatrienoic acids (55), myoendothelial gap junctions (56) and oxidants (57) can also control arterial tone in an endothelial-dependent manner. The specific mechanism of regulation differs depending on how the artery is stimulated, sex, and the location of the artery in the circulatory system. However, arguably the biggest variable that dictates which mechanism is employed is the health state of the artery.

In PAD patients many of the above mechanisms become dysfunctional. For instance, flow mediated dilation (FMD), a surrogate for endothelial function, and commonly associated with endothelial production of NO, is reduced (58). Consistent with this, PAD patient plasma and urine showed decreased BH4, cyclic guanosine monophosphate (cGMP) and decreased NOX's, pointing toward a decrease in endothelial NO bioavailability (59). Ismaeel et al. (60) has since shown a range of increased oxidative stress markers in PAD, proposing these as mechanisms by which NO bioavailability is lost, and is somewhat in agreement with other studies. For example, administration of L-arginine to patients to stimulate NO production, or oxypurinol to decrease oxidative stress (and thus increase NO bioavailability) increased FMD, restored blood flow and decreased patient symptoms (61, 62). However, despite the promise of these short-term studies, longer term clinical trials over 6 months in the NO-PAIN study showed that oral administration of L-arginine did not improve FMD or improve any NO biochemical parameters in patients with intermittent claudication (63). An additional strategy to increase NO bioavailability is to modulate the eNOS enzyme directly. In this regard the β-adrenergic receptors (βARs) may have therapeutic potential, particularly the β₃AR isoform. β₃AR agonists stimulate vasodilation via their ability to modulate eNOS activity and NO production (64, 65). Moreover, we showed that activation of β₃AR restored NO and the redox balance, improving vasodilation and EC function in a mouse model of diabetic PAD (66).

The impact of PAD on other vasodilatory pathways is less investigated but should not be overlooked. Evidence already exists of other pathways such as the COX-dependent regulation of vascular tone that may be disrupted in PAD models (67), and that selective inhibition of COX-2 offers clinical improvement in intermittent claudication (68). Given that the contribution to vascular tone of NO decreases as vessel size decreases (69), future research should also investigate the effects of PAD on non-NO mechanisms of arterial dilation. A summary of these pathways is described in Figure 2.

In hemostasis, injury to blood vessels (e.g., ischemia) activate ECs, which then sprout, migrate, proliferate, and form EC tubules, driven by hypoxia-induced mediators, the most characterized being vascular endothelial growth factor (VEGF). Interestingly, in clinical practice and in animal models of cardiovascular disease, the endogenous angiogenic responses are impaired, particularly with aging (70, 71), in diabetes (72–74) or in dyslipidemia (71, 75, 76). Thus, stimulating angiogenesis in local ischemic tissues could be beneficial in PAD and other vascular diseases. However, to date, all large clinical trials delivering angiogenic factors, including VEGF to people suffering from ischemic diseases such as PAD have shown little benefit (77). Furthermore, the role of angiogenesis in atherosclerosis is conflicting. For example, in a rabbit carotid artery collar model of intimal hyperplasia, adenoviral delivery of VEGF-A, -B, -C and -D increased intimal thickening which was positively correlated with neovascularisation (78). VEGF and other angiogenic molecules including fibroblast growth factor (FGF) were shown to accelerate atherosclerosis in animal models (79, 80), whereas anti-angiogenic therapies reduced atherosclerosis development (81). In contrast, systemic inhibition of the VEGF receptor attenuated established atherosclerosis in high fat diet-fed Apoe^−/− mice (82). Interestingly, plasma concentrations of VEGF-A, but not VEGF receptor-1, are significantly elevated in PAD patients vs. healthy controls (83). In support of this, a significant increase in plasma VEGF was observed in patients with intermittent claudication vs. CLTI, suggesting that VEGF may act as a biomarker or causal factor in disease (83). Indeed, Stehr et al. found increased VEGF levels associated with increased PAD severity (84). What is clear from these studies is that our knowledge of the complex angiogenic pathways and responses to ischemia in PAD is limited.

Other molecules of interest that can promote angiogenesis include TNF-related apoptosis-inducing ligand (TRAIL) and β₃AR. TRAIL is a protein discovered for its ability to selectively kill tumor cells but leave normal cells resistant to its cytotoxic actions (85, 86). Interestingly, in ischemic cardiovascular diseases including PAD, TRAIL levels in the circulation are suppressed (87–90) and in the cardiovascular system TRAIL appears to have homeostatic rather than cytotoxic properties. For example, the presence of TRAIL attenuated atherosclerosis in mice (87, 91), in part by resolving inflammation and improving macrophage function (87), reducing oxidative-stress-induced EC dysfunction (92), and increasing eNOS activity to stimulate intracellular NO production in ECs (93). In the latter study, TRAIL stimulated NO production via a NOX-4-dependent mechanism (93). Furthermore, we and others showed that exogenous TRAIL treatment stimulated in vitro EC processes of angiogenesis (proliferation, migration and tubule formation) (93–95) and promoted stable collateral vessels in the ischemic limb of mice (93). Activation of the β₃AR can also stimulate processes of angiogenesis. For example, the specific β₃AR agonist BRL37344 increased retinal EC proliferation and migration in vitro, whereas stimulation of the β1 isoform, β₁AR had no effect (96). Furthermore, nebivolol-induced angiogenesis in a mouse model of aortic sprouting was abrogated with β₃AR deletion, demonstrating the importance of β₃AR in neoangiogenesis (97). More recently, we showed that administration of CL316,243, a specific β₃AR agonist, stimulated human umbilical vein EC migration and tubule formation in a NOS-dependent manner in vitro, and crucially, CL316,243 improved blood perfusion and angiogenesis in a mouse model of diabetic PAD (98). These findings support TRAIL and β₃AR agonist modulators as promising new therapeutic agents for the treatment of PAD.

Exercise training has positive effects on endothelial function. This is particularly evident in CAD where exercise training improved endothelium-dependent vasodilation that resulted in a 4-fold phosphorylation of eNOS^1,177 from isolated arteries 4 weeks later (102). Increased endothelial function after exercise is also observed in patients with hypertension (103), and in type-1 and type-2 diabetes (104, 105). Furthermore, moderate-intensive exercise (>10 h/week) stimulated coronary collateral blood flow and improved diastolic heart function in CAD patients (106). In this prospective study, 60 patients were randomly assigned to high intensity exercise, moderate intensity exercise or control for 4 weeks. Angiography identified significantly increased coronary flow index in the exercise treated groups (39 and 41%, respectively) vs. control which was associated with increased VO₂ peak (maximal oxygen uptake; measure of aerobic fitness) (106). The authors proposed two mechanisms: recruitment of pre-existing vessels or improved EC function of small intramyocardial vessels (106). Because supervised or home exercise programs are first-line therapy for PAD (101), these mechanisms may also be relevant in limbs. Indeed, exercise training improved brachial artery dilator function in older sedentary females, (107) but these findings are not as apparent in PAD. There is also potential for long-term exercise therapy to improve systemic inflammation since an inverse correlation between exercise, inflammation and plasma CRP levels exist (108). The role of exercise treatment on inflammation in PAD and its effect on EC function is unclear and requires further elucidation.

Phosphodiesterases (PDE) play important role(s) in barrier function by inactivating the messenger cyclic nucleotides cyclic adenosine monophosphate (cAMP) and cGMP and ECs express 5 PDEs, namely, PDE1, PDE2, PDE3, PDE4, and PDE5. Cilostazol is a PDE3 inhibitor and antiplatelet medication used to relieve PAD patients from symptoms of intermittent claudication, which improves walking distance (109), in part by acting as a vasodilator and its ability to stimulate NO release. The most recent Cochrane review, which included 8 placebo-controlled randomized controlled trials involving 2,360 participants with PAD, reported that cilostazol significantly increased maximum walking distance (mean difference 39.6 m. 95% CI 21.8, 57.3; GRADE criteria very low certainty evidence) (110). However, cilostazol was associated with an increased odds of headache which is a common reason for discontinuation. It was suggested that the effects of cilostazol may vary depending on its ability to convert into its active metabolite via the cytochrome P450 system (111). Interestingly, cilostazol may have sex-dependent effects in ECs since female ECs express more Pde3b mRNA than male cells (112). However, no reports of differing responses in men and women have been identified. How cilostazol effects EC function(s) in PAD is not fully established.

Mirabegron is a second generation β₃AR agonist used to treat overactive bladder. The STAR-PAD trial is a Phase II, multicenter, double-blind, randomized, placebo-controlled trial of mirabegron vs. placebo on walking distance in patients with PAD that is currently recruiting by Figtree and colleagues (ACTRN12619000423112) (113). A total of 120 patients aged ≥40 years with stable PAD and intermittent claudication will be randomly assigned (1:1 ratio) to receive either mirabegron (50 mg orally once a day) or matched placebo for 12 weeks. The primary endpoint is change in peak walking distance assessed by a graded treadmill test. Secondary endpoints include: (i) initial claudication distance; (ii) average daily step count and total step count and (iii) functional status and quality of life assessment. Mechanistic sub-studies will examine potential effects of mirabegron on vascular function, including brachial artery FMD, arterial stiffness and angiogenesis. Given that mirabegron is well-tolerated and clinically available for alternative purposes, a positive study is positioned to immediately impact patient care.

Large vessel blockages may not be the only mechanism contributing to PAD pathogenesis. Interestingly, microvascular dysfunction in the limb can increase amputation risk by ~20-fold, even in the absence of large vessel atherosclerosis (7). This is somewhat reminiscent of female patients with CMD who present with dysfunction of the small coronary vessels in the absence of atherosclerosis. These women have worse heart function and blood perfusion (114), with EC dysfunction the primary cause (115). Similar mechanisms may be at play with PAD, given majority of patients do not present with typical symptoms.

Central mechanisms thought to govern CMD include enhanced vasoreactivity at both epicardial and microvascular levels, impaired coronary vasodilator capacity, and increased microvascular resistance; effects of dysfunctional ECs (116). The mainstay of therapies for CMD are β-blockers, statins, calcium channel blockers and angiotensin converting enzyme (ACE)-inhibitors. These are also recommended as secondary prevention for PAD (Table 1). β-blockers and calcium channel blockers reduce severity of anginal symptoms and improve exercise stress test performance (117) and this may be due, in part, to the fact that they also block oxidative stress, improve EC survival (118), reduce EC activation and inflammation, and stimulate eNOS production (119, 120). Furthermore, β-blockers reduce FMD in people with cardiovascular diseases (121) whereas ACE-inhibitors have only showed modest improvement in FMD in patients with CAD (122), even though they modulate survival of ECs (123). Statins not only reduce cholesterol synthesis, but also dampen inflammation. They also do this via their direct effect on ECs. For example, low dose statins improved viability, reduced VCAM-1 and ICAM-1 expression, and atherosclerosis in pre-clinical models (124, 125). They also promoted NO release and repair mechanisms following EC injury (126). Although these medications are recommended for PAD treatment, adherence to these is variable amongst patients (127).

As described earlier, inflammation contributes to atherosclerosis pathophysiology, in part, via endothelial activation, and recruitment of leukocytes to the vessel wall. The Canakinumab Anti-inflammatory Thrombosis Outcome Study (CANTOS), a randomized double-blind, placebo-controlled trial of canakinumab, a monoclonal antibody targeting IL-1β showed that 150 mg of canakinumab reduced recurrent cardiovascular events in patients with stable CAD when compared to placebo (128). Thus, anti-inflammatories could also hold promise for PAD therapy not only in their ability to reduce inflammation, but also since many anti-inflammatories impact EC function(s). For example, anakinra, an inhibitor of IL-1, reduced endothelial dysfunction in diabetic rats; a finding that was associated with decreased NOX and circulating inflammatory cytokines including IL-1β and TNFα (129). Anakinra treatment for 30-days also improved FMD in patients with rheumatoid arthritis (130). Similar findings were also observed with TNFα inhibitors (131). More recently, colchicine has shown considerable promise as a relatively safe, inexpensive dedicated agent which targets inflammation by attenuating NLRP3 activity and IL-1β expression; a recent meta-analysis demonstrating reduced MACE, MI, stroke, and the need for coronary revascularization in patients with coronary disease (132). Data on direct effects of colchicine on ECs is limited, however an older study showed that colchicine reduced the number of E-selectin molecules on the endothelium and subsequent adhesiveness of the ECs to IL-1 or TNFα (133). Although one recent study found no difference in FMD between low-dose colchicine and placebo in patients with CAD (134), another study showed improvement in FMD in patients with a white blood cell count ≥7,500 mm³ (135). Never-the-less, these studies suggest that anti-inflammatory agents could be used to reduce inflammation in PAD and have potential direct effects on EC dysfunction.

As described earlier PAD is associated with dysregulated platelet activation and coagulation, which could precipitate major thrombotic events, observed not only in large vessel disease, but also in small vessels with microthrombi resulting in reduced tissue perfusion. Although current guidelines recommend the use of antiplatelet and antithrombotic medication to reduce the risk of MI or stroke (4, 99–101) there is a significant lack of guideline adherence (136), especially for newly-diagnosed PAD patients (137). Generally speaking, aspirin and clopidogrel are the two most studied antiplatelet medications. Aspirin inhibits COX and subsequent thromboxane A2, which is not only vasoconstrictive, but also activates platelets. Clopidogrel on the other hand prevents platelet activation by blocking the P2Y12 receptor on the surface of the platelet. Both have direct effects on ECs. For example, aspirin protected ECs against oxidized low-density lipoprotein, high glucose, angiotensin II, and H₂O₂-induced injury (138, 139). It also improved impaired acetylcholine-induced vasodilation in patients with atherosclerosis (140) and in pre-clinical models of aging (141). LPS-induced mRNA expression of inflammatory cytokines was attenuated with clopidogrel, associating with improved EC viability, migration, proliferation, and angiogenesis (142). Clopidogrel also prevented endothelial dysfunction in hypertensive rats (143).

Unlike anti-platelet medications, anti-coagulants inhibit the coagulation cascade and the formation of fibrin. An example is Rivaroxaban, a specific inhibitor of factor Xa. In the COMPASS trial which included >27,000 patients with stable CAD or PAD, patients were given low-dose rivaroxaban (5 mg, twice daily) and aspirin (100 mg, once daily), or aspirin alone. Patients assigned to low-dose rivaroxaban plus aspirin had better cardiovascular outcomes after ~2 years, including reduction in the combined risk of cardiovascular death, stroke, and MI (144). However, the risk of major bleeding events increased (144). In a sub-study, rivaroxaban plus aspirin reduced the incidence of major adverse limb events including amputations, when compared to aspirin alone (145). While the risk of major bleeding events was increased, the risk of fatal bleeding was not (145). In the more recent VOYAGER PAD trial (146), >6,500 PAD patients undergoing revascularization received either rivaroxaban (2.5 mg, twice daily) plus aspirin (100 mg, once daily), or aspirin alone for 3 years (147). A significant reduction in ischemic limb events including acute limb ischemia, amputation as well as cardiovascular outcomes (death, MI, stroke) were observed (147). Despite women having higher total cholesterol and greater prevalence in hypertension, diabetes and chronic kidney disease, the net clinical benefit of rivaroxaban plus aspirin was similar with sex, with comparable rates for cardiovascular outcomes and bleeding between men and women (148). The risk of major bleeding was still higher with rivaroxaban treatment.

Interestingly, rivaroxaban also has direct effects on ECs and the endothelium. Rivaroxaban administration improved vasodilation in diabetic wildtype mice, in part by increasing aortic eNOS activity (149). Forearm blood flow was also improved in diabetic patients administered rivaroxaban for 20 weeks, although treatment was associated with higher bleeding events (150). In vitro, rivaroxaban reduced ROS (reactive oxygen species), improved DNA repair (151) and reduced inflammatory gene expression in ECs exposed to hydroxycholesterol (152, 153). Rivaroxaban also stimulated blood flow and increased capillary density in a mouse model of diabetic PAD (154). The same group demonstrated improvement in endothelial progenitor cell migration and senescence, associating with increased eNOS activity in a hyperglycemic environment (154), suggesting that rivaroxaban has pleiotropic functions in ECs. These findings demonstrate that antiplatelet and anticoagulants may improve EC dysfunction in PAD, however additional studies are needed to fully characterize these effects.

It is well established that diabetes mellitus increases the risk of PAD and accelerates atherogenesis. Although it is unclear if intensive glucose control reduces the risk of PAD, studies describe positive outcomes in lower-extremity events including a 31% reduction in risk of amputation with intensive glucose lowering (155). As such, glucose lowering is a recommended PAD guideline pharmacotherapy; its impact on PAD has been reviewed (156). Because hyperglycemia and insulin resistance can facilitate EC dysfunction (157), many glucose-lowering therapies can impact ECs directly. Insulin for example can directly regulate eNOS expression and NO release to cause vasodilation (158), and also via this pathway, inhibit platelet hyperactivity (159). Further, insulin has regenerative and healing capacity in ECs by stimulating angiogenesis (160). Similarly, metformin was shown to improve endothelial-dependent vasodilation in diabetic atherosclerotic mice (161), however, its role in angiogenesis is conflicting (162, 163). More recent studies demonstrate that diabetic patients treated with glucagon-like peptide agonists, sodium-glucose co-transporter-2 inhibitors or in combination, showed improved systolic blood pressure, endothelial glycocalyx thickness and cardiac function (164). This may in part be due to the direct effect of these therapies on EC functions. For example, glucagon-like peptide 1 and sodium-glucose co-transporter-2 inhibitors reduce EC ROS production, reduce adhesion molecule expression and inflammation, improve vasodilation, and stimulate angiogenesis (165, 166). Additional studies are needed to fully comprehend the effect of glucose-lowering agents on EC functions and their cardioprotective effects.

MicroRNAs (miRNAs) are small ~20–25 nucleotide long endogenous non-coding RNA sequences; their main function to regulate protein expression post-transcriptionally. miRNAs are emerging as a biomarker and potential PAD therapeutic. Using next generation genome-wide sequencing, a recent study led by Syed and colleagues identified miRNA-1827 to be significantly upregulated in the blood and plasma of patients with CLTI (167). miRNA-1827 was shown to inhibit cell proliferation and tumor angiogenesis in zebrafish (168) and may in part, contribute to impaired angiogenesis and EC function observed in PAD. miRNA-503 is also upregulated in amputated ischemic limb tissues from diabetic CLTI patients, in ischemic tissues of diabetic mice as well as in ECs exposed to diabetic conditions in vitro (169). Indeed, the authors found that miRNA-504 overexpression inhibited glucose-induced in vitro processes of angiogenesis, whereas inhibition of miRNA-503 improved blood perfusion and increased EC capillary density in diabetic mice following ischemic injury (169). These findings imply that miRNA-503 could be targeted for improving EC function in PAD. Other miRNAs are also altered in PAD patients including miRNA-130a,−27b and−210. Interestingly, miRNA-130a suppression was shown to increase angiogenesis and improve neurological function in ischemic stroke (170) and miRNA-27b inhibited human umbilical vein EC proliferation, migration and tubulogenesis by directly suppressing VEGF-C (171). In contrast, miRNA-210 stimulated pro-angiogenic processes in hypoxia in the brain and in ECs in vitro (172). The mRNA expression of all 3 miRNAs were increased in serum from atherosclerotic obliterans/PAD patients at I-III Fontaine stages (173), however, their role in EC functions in PAD is unclear. miRNAs could be the future in PAD diagnostics and gene therapy [reviewed in (174–176)].

In addition to miRNAs, dysfunction to the endothelium can stimulate the release of endothelial-derived microvesicles (EMVs). These are small vesicles (~0.5–2 μm) released by activated ECs during inflammation to regulate multiple cellular and vascular functions (177), playing a role in immunity, inflammation, and thrombosis (178). They can also carry miRNAs (178, 179). Because of these functions, EMVs are emerging biomarkers with therapeutic potential, particularly in atherosclerosis. For example, EMVs isolated from patients with CAD stimulated permeability and increased the mRNA expression of ICAM-1, VCAM-1, and CCl-2 in ECs in vitro (180). The role of EMVs in PAD is not elucidated, however, similar mechanisms may be at play. Further study is needed to understand their role in PAD.