## 2 Role of ROS in the Origin of Elastin-Derived Peptides  
Elastin occurs naturally in skin, arteries, lung, and other tissues [6]. This protein is characterized by high tolerance to mechanical damage, giving additional resistance of certain tissues. It is also a matrix for cells, especially in the nervous system [17; 18]. The tropoelastin molecule is the building-block of elastin, which makes this protein nearly insoluble in water and responsible for its unique properties [19; 20]. It has been observed that the half-life of elastin is over 70 years [21]. However, an intensified degradation process of this protein is observed during aging [22]. Hence, it is difficult to study the direct effects of elastin on the entire organism, especially on ROS production. However, in the 1950s, the first in vitro hydrolyzation of elastin was performed with the use of potassium hydroxide (KOH), resulting in \(\kappa\)-elastin production. Subsequently, the degradation of elastin by oxalic acid (C\({}_{2}\)H\({}_{2}\)O\({}_{4}\)), which yields an \(\alpha\)-elastin molecule, was developed [23]. Thus, the next discoveries in this field were focused on characterization of the obtained molecules as products of elastin degradation called elastin-derived peptides (EDPs). It has been observed that the peptides with the Gly-x-x-Pro-Gly (GxxPG) amino sequence were repeated many times in the EDP structure, which defines the conformation of elastin [24]. The most common sequence, i.e., the Val-Gly-Val-Ala-Pro-Gly (VGVAPG) hexapeptide, has been found in the EDP group [25]. Literature highlights three main causes (types) of elastin degradation: chemical, enzymatic, and ROS-dependent (Figure 1). The first type can be achieved with the use of potassium hydroxide (KOH) or oxalic acid (C\({}_{2}\)H\({}_{2}\)O\({}_{4}\)), which are not present in the organism but are used only to prepare EDPs in vitro given their physicochemical properties. The second type of elastin degradative factors is represented by various proteinases, which act both in vitro and in vivo. To date, three groups of proteolytic enzymes acting as elastases have been described [26]. Serine proteases released from neutrophils or macrophages act as chymotrypsin-like proteins. They are able to degrade the whole elastin molecule or pre-degraded protein, e.g., proteinase 3, neutrophil elastases, and cathepsin G [27]. Another group comprises cysteine proteases such as cathepsin K (CatK), L, S, and V, which naturally occur in lysosomes [28]. The third group of proteases capable of hydrolyzing elastin are metalloproteinases (MMPs), which require metal ions for their hydrolyzing activity, e.g., MMP-2, -7, -9, and -12 [26]. The last type of elastin degradation is the ROS-dependent mechanism. To date, it has been described that tropoelastin is sensitive to ROS [29]. It has been shown that ROS generated by ultraviolet A (UV-A) and hematoporphyrin rapidly degraded tropoelastin within 5 min [29]. Treatment of tropoelastin with copper sulfate/ascorbic acid resulted in  
Figure 1: Types of elastin degradation factors: chemicals, ROS-dependent, and enzyme-dependent. ROS—reactive oxygen species, MMPs—matrix metalloproteinases, EDPs—elastin derived peptides. degradation of tropoelastin producing fragments of molecular weight 45, 30, and 10 kDa within 30 min. The degradation of tropoelastin was partially blocked by the addition of mannitol, which is a well-described radical scavenger [29; 30]. ROS generation induced by the xanthine-xanthine oxidase system caused degradation of tropoelastin within 6 h. The degradation was blocked by the antioxidant enzyme catalase (CAT), which is a ROS scavenging enzyme, proving the engagement of free radicals in the elastin degradation process. ROS generated by copper-ascorbate seems to be unique in that it cleaves relatively specific sites of the tropoelastin molecule. Thus, ROS may play a degradative role in elastin metabolism, which may cause elastolytic changes or deposition of fragmented elastic fibers in photoaged skin or age-related elastolytic disorders [29].