In our study, patients with significant lower-limb PAD requiring endovascular revascularization appeared with significantly higher circulating levels of hsCRP, OPN and OPG compared to age- and sex-matched individuals without CVD. Multivariate analysis confirmed an independent association of those biomarkers with the presence of PAD. Among PAD patients, those developing MACE during the 12-months follow-up, had even higher hsCRP, OPN and OPG circulating levels, than non-MACE group, but we failed to detect their prognostic value in regression analysis.
Osteoprotegerin and atherosclerotic diseases
Regarding OPG, our results are in line with most previous studies. Among 98 patients with diabetes with or without PAD, OPG was independently associated with > twofold risk of PAD (odds ratio- OR:2.26) . Within type 2 diabetes mellitus (T2DM) population, higher OPG levels have been found in PADG compared to COG (p < 0.001) . Notably, O’ Sullivan et al.  confirmed higher serum OPG in PAD patients than controls, regardless of the co-existence of T2DM. The association between high OPG and PAD seems to be also independent of race, in a large-scale study including 1291 African–Americans and 1152 non-Hispanic whites, while a strong and consistent positive association has also been reported in patients in peritoneal dialysis . On the other hand, a small number of studies has failed to demonstrate such association. In details, a study enrolling T2DM patients without known CVD, demonstrated significantly increased OPG levels in patients with carotid and peripheral arterial disease compared to controls, but this association remained significant only for carotid disease after adjustment for age, glycosylated hemoglobin (HbA1c) and urine albumin-to-creatinine ratio . Ziegler et al. , found no difference in OPG concentrations between 67 patients undergoing PTA of PAD and 94 age-matched healthy controls. However, in order to properly interpret these seemingly contradicting results, it must be noted several drawbacks in those studies, like the absence of data about ABI measurement, glycaemic status or CVD history which questioned the severity of PAD and blurred its association with OPG.
In our study, although elevated baseline OPG levels were associated with the incidence of cardiovascular complications or restenosis requiring re-revascularisation among PAD patients; this association was not confirmed in multiple regression analysis. Previous research data favour a causal relationship between OPG and MACE, but the evidence is not robust. Similarly, Biscetti et al.  showed the association between baseline levels of various inflammatory cytokines (OPG among them) and failure of lower extremity endovascular revascularisation in patients with diabetes, PAD and chronic limb-threatening ischemia (CLTI). A significant linear association between the lowest and highest quartiles of OPG and the risk of major adverse limb events (MALE: acute limb ischemia, major vascular amputations and urgent limb revascularisation) as well as the risk of MACE was found even after adjusting for other risk factors in multivariate models. The prognostic value of OPG has been implicated in patients with stable angina pectoris, but independent effects were limited to levels above the 90th percentile , while higher OPG levels have also been associated with higher incidence of internal carotid stenosis in diabetics compared to those without stenosis . In a recent meta-analysis of 9 studies including 26,442 participants of general population, those in the top third of OPG concentration had higher combined risk ratio for CVD (1.83), CAD (1.72), and stroke (1.58), compared to individuals at the bottom third of OPG concentration, implicating its predictive power . However, when the meta-analysis focused only on high-risk populations, namely diabetes mellitus, chronic kidney disease (CKD), pre-existing heart disease or recent acute coronary syndromes, this predictive power was significantly attenuated . Therefore, those inconsistent results could be explained by the heterogeneity of concomitant treatment regimens; for instance, some antidiabetic agents and statins are well-known to affect circulating OPG levels [30, 31]. Notably, within our PADG, a small subgroup of 10 statin-free patients had higher OPG levels compared to statin-treated PAD patients. In addition, OPG levels vary among different diseases, such as PAD, diabetes and CKD. We cannot exclude that its association with MACE can be attenuated also because of the dominance of other risk factors more relevant to CVD risk, such as metabolic syndrome, smoking or family history. In the majority of previous studies, bone status was not assessed, which could have seriously affected OPG levels. Hence, the prognostic value of OPG remains under investigation.
Osteoprotegerin and atherosclerotic pathophysiological mechanisms
Whether OPG mediates or protects atherosclerosis is unclear. On the one hand, OPG acts as a soluble decoy receptor for the receptor activator of nuclear factor-B ligand (RANK-L) and the TNF-related apoptosis inducing ligand (TRAIL) . It has been postulated a compensatory, yet often failed, self-defence response of OPG to atherogenesis. On the other hand, OPG can exert pro-inflammatory effects; by increasing macrophage infiltration and leukocyte adhesion to endothelial cells, and sensitizing them to the effects of TNF-alpha via upregulation of angiopoietin-2 [33, 34]. RANKL promotes osteogenic differentiation of vascular smooth muscle cell and stimulates the release of various pro-inflammatory cytokines, such as matrix metalloproteinase (MMP)-9, while TRAIL is a potent activator of apoptosis. Therefore, the action of OPG exerts an anti-inflammatory, anti-apoptotic and anti-calcifying effect [6, 35]. In addition, by blocking RANK-L, OPG reduces nitric oxide synthase production and leads to decreased vascular dilatation and endothelial dysfunction . Finally, a role in plaque destabilization has been implied, however, the study data remain rather conflicting .
Osteopontin and atherosclerotic diseases
Regarding the relationship between OPN and PAD, literature data are scarce, but they generally favour a positive association. Recently, a cross-sectional study enrolling 70 individuals with T2DM and 66 controls, showed significantly higher plasma OPN levels in PAD patients compared to PAD-free counterparts (p < 0.001), regardless of their glycaemic status . In addition, OPN was negatively associated with ABI (Spearman’s r = − 0.245 in the whole sample), indicating a positive association with PAD severity. It must be noted, that a range of OPN levels that could be considered normal has not yet been determined, with median values reported among healthy individuals ranging between 20.25 and 55.1 ng/mL.
A significant amount of evidence exists regarding the association of OPN with MACE, although these data derived mainly from CAD or stroke populations. In a sub-analysis of the PEACE trial, where 3567 patients with stable CAD were studied, OPN was significantly associated with the composite primary endpoint of cardiovascular death, non-fatal myocardial infarction and hospitalization for heart failure, even after adjustment for relevant co-variates . However, a secondary analysis showed that this association was predominantly driven by the hospitalization for heart failure. In PAD patients, Lin et al.  reported high OPN levels as strong predictors of all-cause death, with the optimal cutoff concentration for predicting mortality being 126 ng/ml . In agreement, we observed a positive association between OPN and PAD, which was lost after multiple logistic regression analysis. An even stronger association between OPN and MACE was revealed in a similar population by Georgiadou et al. (HR: 2.88) for OPN levels > 55 ng/ml , but those levels were considerably lower than the median values in our cohort and the study by Lin et al. . OPN has also been associated with restenosis after coronary revascularization , but this has not been a consistent finding . In 80 patients under chronic hemodialysis, OPN levels were associated with the severity of carotid stenosis , and in a large cohort of 3545 ischemic stroke patients, elevated OPN at baseline was a risk factor for adverse clinical outcomes at 1 year after ischemic stroke, rendering OPN a possible prognostic biomarker . Overall, the existing data, predominantly from CAD studies and to a lesser extent from PAD trials, are controversial concerning the prognostic value of OPN. Methodological factors, such as sample size and follow-up duration, or the concomitant use of lipid-lowering or antidiabetic agents are possible explanations for those inconsistent results.
Osteopontin and atherosclerotic pathophysiological mechanisms
OPN mediates its vascular effects through complex pathways. Although hardly detectable under physiological conditions, OPN expression is often increased 20–50-fold in response to acute ischemic events, being a key player in the immune and inflammatory response . More specifically, OPN promotes chemotaxis and recruitment of macrophages, as well as smooth muscle cell proliferation, thus contributing to increased inflammation and post-ischemic neovascularization. States of chronic, low-grade inflammation and endothelial dysfunction, such as hyperglycemia, hypoxia or dyslipidemia, have also been shown to increase OPN expression in VSMCs, which in turn exacerbates oxidative stress and inflammation, leading to a vicious cycle. Apart from inflammation; increased levels of OPN have been found within human atherosclerotic plaques from the aorta, carotid and coronary arteries . In mouse models, OPN overexpression significantly increased atherosclerotic lesions , while depletion of one or both OPN genes attenuated angiotensin II–accelerated atherosclerosis  and treatment with liraglutide significantly attenuated OPN expression in a rat model of metabolic syndrome . On the other hand, OPN is a potent inhibitor of vascular calcification, an effect which may be considered vasculoprotective. However, the anti-calcifying effect of OPN is not consistent, but it actually relies heavily on its phosphorylation status .
Osteoprotegerin and osteopontin and chronic kidney disease
In our study, we excluded patients with severe CKD, however 23% of our PAD patients appeared with mild or moderate (stages 2–4) CKD. Regarding the increase of OPG and OPN levels along renal function worsening , we cannot rule out the contribution of renal impairment to the higher serum levels of those biomarkers in PADG than COG. On the other hand, a significant difference in OPG and OPN serum levels persisted between MACE and non-MACE groups, despite the absence of significant difference in kidney function. Moreover, univariate and multivariate analysis did not show any significant correlation of creatinine and GFR with OPG and OPN. Hence, the interplay of CKD with OPG, OPN is not clear in our PAD cohort and its impact on prognosis seems undetermined.
The relatively small sample size and the short follow-up duration are among the most common limitations of our study. Furthermore, no assessment of bone status was conducted. Another important drawback is the fact that there is a wide variety of factors affecting circulating levels of biomarkers, which prevented the demonstration of a cause-effect relationship between examined biomarkers and clinical outcomes. Moreover, the vast majority of our patients was already on optimal pharmaceutical therapy, which might have influenced the levels of used biomarkers and clinical outcomes, undermining the predictive power of biomarkers. Another limitation was the relatively high smoking rate among PAD patients, driving the restenosis rate. However, this is common in clinical practice and reflects an unavoidable situation.