The first result of our study is that sortilin levels correlate statistically significant with ABI and are higher in patients with lower ABI. Unsurprisingly, patients with a more severe PAD, at time of enrollment, had higher sortilin levels than those with less severe disease.
The management of cardiovascular complications of T2DM is an unresolved problem. In particular, MACE and MALE in T2DM patients with PAD and CLTI are very frequent and are caused by pathological mechanisms, which can be relevant from a diagnostic point of view or considered an important therapeutic objective. For example, cholesterol, in particular LDL-C, constitutes both a risk factor for atherosclerotic pathologies and a risk stratification biomarker, but it is also an important objective of medical therapy.
Among the molecules involved in LDL-C pathways, sortilin is a promising biomarker. In fact, sortilin is involved in LDL-C trafficking. On the one hand, a direct relationship between intrahepatic sortilin concentration and plasma LDL-C levels exists, both through an ApoB100-mediated mechanism and increased secretion of proprotein convertase subtilisin/kexin type 9 (PCSK9). On the other hand, serum levels of sortilin may have a direct role in the atherosclerotic plaque formation, with an independent LDL-C mechanism. In fact, the action of sortilin in immune cells, including macrophages, can worsen the development of atherosclerosis, with mechanisms not yet fully described, and in any case not exclusively dependent on the uptake of LDL-C[13, 19].
The role of sortilin in LDL metabolism is still unclear. Hepatic sortilin increases the clearance of LDL-C and reduces the secretion of very-LDL (VLDL); in contrast, studies performed in transgenic mouse models have shown the opposite effect[25, 28]. Therefore, specifically the role of circulating sortilin remains unclear or more precisely, if sortilin has a direct role on lipid metabolism and on the formation of atherosclerotic plaque, or if it represents an epiphenomenon useful as biomarker of cardiovascular risks in selected patients. In fact, substantial difference between data obtained on diabetic and non-diabetic populations exists. Notably, in a relatively small cohort of patients, sortilin levels were reduced in the diabetic compared to non-diabetic patients. Furthermore, the reduced levels were also associated with a worse metabolic profile. However, it should be noted that the potentially confounding and influencing effects of some drugs, such as metformin, have not been studied in this report.
Finally, a further element worthy of analysis is centered on the substantial difference existing between the two pathological conditions, CAD and PAD. In fact, an important distinction has been described in terms of comorbidity between T2DM patients with CAD or CLTI requiring revascularization. This discrepancy could contribute to data heterogeneity, even conflicting data, available in literature and contributed to define the objective of our study.
Starting from these biological supports, a detrimental role of sortilin for diabetic patient has been previously confirmed[20, 31]. Furthermore, sortilin levels correlate with the presence of PAD and PAD severity in a cohort of diabetic patients. Here we found that sortilin levels at baseline are higher in patients with lower ABI. These results are in line with previous data and support the conclusion that an association exists between serum levels of sortilin serum levels and PAD severity in T2DM patient. However, the most relevant result of our research is that in the group of patients with higher sortilin levels at baseline, MACE incidence was more frequent, during the 12-months follow-up, compared with the lower sortilin level group.
The finding is strengthened and confirmed for several reasons. First, the different components of the composite outcome, death, CAD and CVD, showed a similar relationship with baseline serum protein levels. In addition, the ROC curve demonstrated sortilin’s predictive capability of MACE. Finally, multivariate analysis adjusted for traditional cardiovascular risk factors confirmed sortilin’s independent relationship with MACE, death, CAD and CVD. Several possible explanations underlie our data. Sortilin can directly induce a robust inflammatory response[19, 32], because the protein is involved in the interferon-γ, interleukin-6 and toll-like receptor pathways, leading to an important activation of macrophages[19, 32, 33]. In addition to the pro-atherogenic effect, sortilin could induce atherosclerotic plaque instability through inflammatory milieu spread. Plaque formation and subsequent instability could determine the incidence of MACE after LER. Indeed, this suggestion is supported by previous data showing an association between sortilin and CAD, both in diabetic and non-diabetic populations[11, 26].
Sortilin levels, to stratify the initial risk of MACE after LER, are an important new biomarker for clinicians who follow T2DM patients. In fact, up to now, it is impossible to predict the likelihood that patients experience MACE hampering personalized follow-up. Our findings give hope that increasing surveillance after LER in patients with high baseline sortilin levels becomes achievable. Considering also the relationship between sortilin and PCSK9, it is reasonable to use early PCSK9 inhibitors in patients with high sortilin levels.
The secondary objective of this study was to explore the association between the basal levels of sortilin and the development of MALE after LER showing that the group of patients with higher sortilin levels had a higher incidence of MALE during the follow-up. Several T2DM patients with PAD and CLTI developed arterial stenosis recurrence after LER and required frequent endovascular treatments. Numerous studies have been carried out which attempted to identify predictive indicators for MALE after LER. Some encouraging data have been drawn from the prospective assessment of selected inflammatory cytokines. However, definitive biomarkers are not established yet. Sortilin could be included to the inflammatory panel used to stratify cardiovascular risk, since it is involved in the inflammatory processes correlated to atherosclerosis. Considering it as a biomarker of cardiovascular events, the ROC analysis has highlighted that sortilin has a remarkable predictive capacity of MACE and MALE in our population. The AUC resulting from our analysis, in particular for the MACE outcome, is significant and is not prejudiced by the cutoff values, which are usually chosen with conventional approaches of sensitivity and specificity.
Our study has several limitations. First, the patient cohort is relatively small and the results need to be confirmed on a larger number of patients. The small sample size also depends on the restrictive inclusion criteria, which, on the other hand, represent a strength of the study. A further limitation is that few events have occurred in patients with sortilin levels in the lower tertile; therefore, it is not possible to assert with certainty that reduced sortilin levels are protective as higher levels are predictive of cardiovascular events. Furthermore, we have included only statin-free patients, to reduce bias on sortilin levels correlated to different therapies. However, after LER, all patients started lipid-lowering therapy to reach the LDL-C target suggested by international guidelines, which might have influenced circulating sortilin levels during follow-up—a factor not evaluate in our study. Furthermore, considering the extent of the follow-up, it is possible that different lipid-lowering therapies have led to different effects on sortilin levels. However, it is unlikely that they played a direct role on the incidence of MACE, considering that the study population had moderate baseline LDL-C levels. Furthermore, sortilin has been shown to affect the PCSK9 pathway with a mechanism independent of statin therapy. Importantly, our primary objective was to study the relationship between baseline values and MACE development after LER, to identify a possible risk stratification biomarker. In this sense, our results are statistically relevant. A further limitation of our study is that we have not studied the relationship between the initial arterial lesion and the occurrence of subsequent MALE. Therefore, we excluded patients with primary LER failure from the study to decrease bias. Moreover, we did not stratify patients based on MALE type, also given the small number of patients.
It is not clear where sortilin can originate in our patient population. Sortilin derives from pro-sortilin, which is converted into the Golgi apparatus of many cell types. This protein has been found abundantly in the central nervous system, but also in the spinal cord, skeletal muscle, testes, heart, placenta, pancreas, prostate, and small intestine. Previous data suggest that hepatic sortilin is associated with a less dangerous lipid profile[34, 35]; however, investigations on animal models have shown that plasma sortilin could play a harmful role on atherosclerosis development, by non-LDL-C-dependent mechanisms. Nevertheless, there are no convincing data on the source of sortilin in serum. Some reports have shown that increased sortilin levels are associated with depression, increased alcohol intake and BMI. In this scenario, it could be speculated that sortilin may worsen cardiovascular outcomes also by acting on other risk factors. However, we have not studied depression in our patient population, we cannot confirm this hypothesis and further studies are necessary and warranted.