*1.1. Mechanisms of PAD*

Diabetes mellitus, especially if not properly treated, can cause damage to different organs and tissues of the human body. Microvascular complications include retinopathy, neuropathy and nephropathy, while macrovascular complications include cardiovascular diseases, stroke and peripheral vascular disease that can lead to bruising or lesions that do not heal, gangrene and, in severe cases, limb amputation. Peripheral artery disease (PAD), also called peripheral vascular disease, is caused by the narrowing of the lumen of blood vessels that carry blood to the arms, legs, stomach and kidneys. In people affected by DM, the risk of PAD increases with age, with the duration of diabetes and in the presence of neuropathy. Other factors associated with cardiovascular diseases, such as high C-reactive protein and homocysteine levels, are also associated with an increased risk of PAD [13,14]. Peripheral arteriopathy is characterized by two types of symptoms: intermittent claudication (or intermittent pain, which may occur during exercise or walking, but resolves with rest) and pain at rest (which is caused by chronic hypoxia/ischemia in the limb, with inadequate blood flow to the affected limb). This pathology is an important risk factor for lower-extremity amputation. Unfortunately, its diagnosis is often made too late and occurs when symptoms are marked, delaying a preventive treatment [15]. Insulin resistance, commonly manifested in type 2 diabetes mellitus (T2DM), is a consequence of several risk factors represented by obesity, sedentary behavior and aging; therefore, hyperglycemia, diabetes, hypertension and dyslipidemia are concomitant diseases often observed in patients affected by PAD [10]. In patients with DM, the diagnosis of symptomatic PAD is approximately twice as high as in patients not affected by diabetes [16]. Recently, Lilja et al. showed that men with DM developed symptomatic PAD more frequently compared to women (15.5% vs. 8.9%) [17], even if not all studies have observed significant differences in the prevalence of PAD between sexes.

Several molecular pathways are involved in the pathogenesis of arterial insufficiency, such as (i) endothelial dysfunction, e.g., the presence of increased plasma levels of asymmetric dimethylarginine (ADMA) is associated with endothelial vasodilator dysfunction, causing arterial stiffness and a reduction in NO• production [18,19]; (ii) endoplasmic reticulum (ER) dysfunction, e.g., vascular endothelial cells of hyperglycemic subjects are characterized by an altered, rough endoplasmic reticulum and protein folding [20,21]; (iii) promotion of inflammation by secretion of cytokines, such as TNF–α, IL–1, IL–6 and IL–8, and chemotactic stimulus for monocytes and macrophages [22]; (iv) mitochondrial dysfunction and increased oxidative stress-induced damage, also linked to the activation of the transcription nuclear factor–κB (NF–κB) [23,24]; and (v) interaction between advanced glycation end-products (AGEs) and their receptors, causing inflammation and endothelial dysfunction [25,26].

The differences in endothelium function between women and men have been reported in various studies. The beneficial vascular effects of estrogens are related to the modification of the functional state of the endothelium [27]. The impaired endothelium-dependent vasodilation of the coronary or peripheral vasculature is positively correlated with an increased risk of cardiovascular events and is an independent predictor of vascular morbidity and mortality.

#### *1.2. Role of AGEs*

In conditions of persistent hyperglycemia, molecular rearrangements occur between tissue proteins and glucose or other reducing carbohydrates, leading to the irreversible formation of AGEs, which significantly contribute to the development of complications associated with DM, including PAD [28]. It has also been observed that the accumulation of AGEs worsens endothelial function [29,30].

The formation of AGEs begins with the non-enzymatic glycation of free amino groups by sugars and aldehydes, which leads to a succession of rearrangements of intermediate compounds and, finally, to irreversibly bound products known as AGEs [24]. Glycation and oxidative stress are intricately linked, and both phenomena are referred to as glycoxidation [28]. Persistent hyperglycemia and oxidative stress accelerate the formation of AGEs [31]. These have mainly been detected in long-lived proteins, with post-translational AGE modifications mainly occurring at the side-chain amino groups of the lysine and arginine residues. Glycation causes the irreversible modification of the protein structure and consequent loss of functionality, leading to detrimental effects in tissues, i.e., vasculature. Glycated proteins become less prone to proteolysis as a consequence, e.g., the accumulation of glycated collagen causes the thickening of blood vessels. Recently, authors showed that AGEs can cause the apoptosis of endothelial progenitor cells via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and promote atherosclerosis [32]. Nucleotides and lipids are also possible targets of glycation, causing the DNA mutation and modification of the integrity of the cell membrane, triggering cell death. Thus, it can be believed that compounds that counteract the effect of macromolecule damage (glycation-protective effects) can slow down the progression of vascular damage in the legs and, furthermore, also in other districts, such as the brain, protecting the health of humans [33]. Recently, several natural compounds, i.e., flavonoids and more generally polyphenols, have received attention for their endothelial protective effects [26,34].
