In this study we have evaluated the association between serum HMGB-1 levels and cardiovascular outcomes after LER in a cohort of diabetic patients with PAD and CLTI, showing an association between higher serum HMGB-1 levels and MACE and MALE during the follow-up period after revascularization.
Despite best medical therapy and a multidisciplinary approach, prevention of cardiovascular complications in T2DM patients with PAD after LER already represents an unmet need. Even though similar baseline characteristics of the population receiving LER, some patients develop no complications, while others have poor outcomes even shortly after the endovascular procedure (34).
Therefore, in addition to traditional cardiovascular risk factors, identifying predictors of adverse outcomes after LER is necessary to prevent cardiovascular events.
Inflammation is a cornerstone of atherosclerosis progression (42), and anti-inflammatory therapy has been demonstrated to have an adjuvant role in the secondary prevention of cardiovascular events (43–45).
The chronic inflammatory state associated with diabetes mellitus plays a key role in macrovascular and microvascular complications in diabetic patients (18, 46). We previously showed in a cohort of 299 diabetic patients with PAD that baseline OPG, TNF-α, IL-6, and CRP levels were associated with adverse cardiovascular outcomes after LER (17). In addition, Bleda and colleagues found associations between CRP and fibrinogen levels with mortality and cardiovascular outcomes at baseline and after LER (16).
HMGB-1 is a ubiquitous protein that acts as a pro-inflammatory cytokine in the extracellular space. It plays a crucial role in the progression of atherosclerosis and the development of cardiovascular diseases (18). Once bound to its receptors (RAGE and TLRs), HMBG-1 activates various signaling pathways, including nuclear factor-kB (NF-kB), extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38MAPK), c-Jun N-terminal kinase (JNK), and myeloid differentiation factor-88 (MyD88), which promote oxidative stress and enhance the secretion of various cytokines, including TNF-α, IL-6 and IL-1, growth factors and adhesion molecules, contributing to endothelial dysfunction and vascular injury (18). HMGB-1 has been shown also to promote vascular endothelial growth factor (VEGF) dependent angiogenesis in animal models of limb ischemia, suggesting a role of HMGB-1 in tissue repair (47, 48).
Higher HMGB-1 levels are associated with poor outcomes after myocardial infarction (MI) (49–51). Specifically, Kohno and colleagues showed that patients with ST-elevation myocardial infarction (STEMI) had higher serum HMGB-1 levels at baseline and 12 hours after the event than patients with chronic stable angina, and this was associated with adverse cardiovascular outcomes, in particular pump failure, cardiac rupture and cardiac death (49). SØrensen and colleagues showed an association between higher HMGB-1 levels and increased mortality after STEMI (50). Hashimoto and colleagues found similar results in a cohort of patients with unstable angina and non-ST-segment elevation myocardial infarction (NSTEMI), suggesting an association between higher HMGB-1 levels and risk of cardiovascular death (51). We have previously shown that elevated serum HMGB-1 levels are associated with the presence and the severity of PAD in diabetic patients, compared with diabetic patients without PAD (33); however, an association between HMGB-1 and poor cardiovascular and limb outcomes after LER has not been demonstrated.
In the present study, we found that serum HMGB-1 levels in diabetic patients with CLTI were associated with MACE after LER. In particular, patients with higher levels of HMGB-1 had a higher risk of myocardial infarction, stroke and death from cardiovascular diseases. Smoking status and higher triglycerides levels were also associated with MACE, in line with previous findings (52). In addition, non-smokers and former smokers were less likely to develop a MACE.
The relationship between HMGB-1 levels and MACE persisted even after adjusting for traditional cardiovascular risk factors such as age, BMI, smoking habits, hypertension, blood lipids, glycemic control and renal function, confirming HMGB-1 as an independent risk factor. Interestingly, ABI was also associated with MACE in the multivariable analysis, supporting the results of previous work by Mendes-Pinto and colleagues (53). Surprisingly, traditional cardiovascular risk factors did not appear to play a role in cardiovascular complications after LER. This result may be due to the small sample size of our cohort, perhaps the intrinsic advanced disease bias of our cohort, and the short follow-up time. However, this finding may also be representative of a distinctive pro-inflammatory state after peripheral vascular revascularization in which HMGB-1 may have key role in clinical outcome prediction. Specifically, we elaborated a ROC curve that included only HMGB-1, confirming the role of HMGB-1 in predicting MACE after LER, and a second ROC curve that included serum HMGB-1 levels and traditional cardiovascular risk factors. Comparing these ROC curves, we found that the model with HMGB-1 and traditional cardiovascular risk factors improved the prediction of MACE.
An additional novelty of our study is that we found a cut-off value for HMGB-1 that could predict MACE survival after endovascular revascularization. HMGB-1 levels above 6.905 ng/mL predict a high risk of death after a MACE with a good sensitivity (69.14%) and specificity (78.33%).
Another finding of our study was that higher HMGB-1 levels were even associated with MALE after LER. Compared with patients without MALE, patients with MALE were active smokers, were on insulin and had higher levels of total cholesterol and triglycerides. Conversely, non-smokers were less likely to develop a MALE.
The association between triglycerides and other cardiovascular risk factors with MALE was attenuated in our multivariable logistic regression after adjusting for HMGB-1, which was confirmed to be an independent risk factor for MALE, along with use of insulin, ABI, glycated hemoglobin and total cholesterol levels, consistent with earlier evidences (54, 55). Finally, the role of HMGB-1 as a predictor of MALE was demonstrated by a ROC curve.
Various explanations could support our results. In fact, an important pro-atherogenic role of HMGB-1 has been demonstrated in the literature (56–58). HMGB-1 acts by regulating various inflammatory factors and immune cells; it also promotes the recruitment of vascular smooth muscle cells (VSMCs) and foam cells in atherosclerotic lesions (56). VSMCs themselves enhance HMGB-1 secretion in an autocrine loop, which may lead to neointimal hyperplasia responsible of restenosis after angioplasty (56). Furthermore, Wang and colleagues demonstrated that LPS/ATP activation of NLRP3 inflammasome stimulates HMGB-1 release leading to cholesterol accumulation in VSMCs and foam cells formation during atherosclerosis, through the downregulation of LXRa and ABCA1 expression (58). HMGB-1 is even responsible of atherosclerotic plaque vulnerability, possibly through VSMCs secretion of matrix metalloproteinase (MMP), enzymes responsible for matrix degradation (32, 59).
Interestingly, high expression of HMGB-1 was found in platelet-rich coronary thrombus from MI patients (60), suggesting that activated platelet-derived HMGB-1 may represent a link between atherothrombosis and inflammation (61). In fact, activated platelets have been found to be the major source of HMGB-1 in thrombi, and activated platelet-derived HMGB-1 has been shown to play a key role in small vessels thrombosis, by promoting platelet aggregation, platelet granule secretion, and immune cells recruitment (61).
The evidences discussed support our findings and indeed HMGB-1 may be an essential mediator of acute cardiovascular outcome after LER and in acute limb adverse events after revascularization.
However, this study has several limitations. First, this is a single-centre study based only on the Italian population and the cohort analyzed was relatively small. Therefore, the results cannot be applied to other ethnic groups and must be validated in larger patient groups. The small sample size may be the reason why the multivariable analysis failed to capture the effects of traditional risk factors on MACE and MALE, along with the short follow-up time of observation.
Furthermore, we did not assess levels of other cytokines, so we could not conclude whether the association between HMGB-1 and poor adverse events after LER remained significant after adjusting for other inflammatory parameters.
Another limitation of the study is that we did not evaluate changes in HMGB-1 levels during follow-up after LER and their impact on cardiovascular adverse events. However, our goal was to identify a biomarker that at baseline could predict poor outcome after revascularization.