**1. Introduction**

Snakebite accidents in humans are a severe global health problem that mainly affects the poor and economically active population, causing significant social issues [1,2]. The World Health Organization (WHO) estimates that approximately 2.7 million snakebite accidents occur annually worldwide. As a result of these accidents, an estimated 81,000 to 138,000 deaths occur, and 400,000 survivors experience permanent sequelae [3,4].

In Brazil, an average of 29,000 snakebites occur annually, resulting in approximately 120 deaths/year [5], in addition to underreported cases. Of these 29,000 envenomations, approximately 90% are caused by snakes of the *Bothrops* genus [6].

In *Bothrops* snakebites, severe tissue loss at the site of the bite is observed as a result of hemorrhage and an exacerbated local inflammatory response induced by the venom.

**Citation:** Zychar, B.C.; Clissa, P.B.; Carvalho, E.; Alves, A.S.; Baldo, C.; Faquim-Mauro, E.L.; Gonçalves, L.R.C. Modulation of Adhesion Molecules Expression by Different Metalloproteases Isolated from *Bothrops* Snakes. *Toxins* **2021**, *13*, 803. https://doi.org/10.3390/ toxins13110803

Received: 18 October 2021 Accepted: 9 November 2021 Published: 15 November 2021

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Among the components present in these venoms, metalloproteases are considered to be some of the main factors responsible for local inflammation and necrosis [7–9].

Some of the most abundant proteins found in *Bothrops* venoms are snake venom metalloproteinases (SVMPs), zinc-dependent proteinases that belong to the reprolysin subfamily [10]. Analysis of the gene expression in the venom gland of *Bothrops jararaca* (*Bj*) snakes showed that more than 50% of transcribed genes are SVMPs [10]. SVMPs are classified as PI to PIII, according to the presence or absence of disintegrin and cysteinerich domains together with a typical metalloproteinase domain, at least in the precursor molecule form [11].

Several hemorrhagic metalloproteases have been isolated from *Bothrops jararaca* venom (*Bj*V) [12–14]. One of the best characterized proteins is jararhagin (Jar), a protein with a molecular weight of 52 kDa that contains PI, metalloproteinase, ECD-disintegrin (ECD: Glu-Cys-Asp) and cysteine-rich domains [14], which are characteristics of an SVMP of the PIII class. In general, the toxins belonging to this class are highly hemorrhagic, and this activity depends on the metalloproteinase domain. However, ECD-disintegrin and cysteine-rich domains have also been shown to be important for the biological functions of these toxins [15–17].

Two forms of Jar are present in *Bj*V: the molecule with the three domains, as described above, and jararhagin-C (Jar-C), a nonhemorrhagic molecule containing only the ECDdisintegrin and cysteine-rich domains produced by the proteolytic cleavage of Jar [18]. The absence of the catalytic site in this toxin does not prevent the inflammatory activity of the toxin. Jar-C triggers the local release of cytokines and induces alterations in leukocyteendothelium interactions, similar to the inflammatory response caused by Jar [9,19].

PI-class SVMPs contain only a catalytic domain, have a molecular mass ranging from 20 to 30 kDa, possess fibrin(ogeno)lytic activity, and mostly present weak hemorrhagic activity. Metalloproteases isolated from *Bothrops neuwiedi* venoms (*Bn*V), such as neuwiedase (PI-class SVMP), degrade fibrinogen, fibrin, type I collagen, fibronectin, and laminin and induce inflammatory reactions [20].

Two other PI-SVMPs analogous to neuwiedase were isolated from *Bn*V: BnP1 and BnP2. However, only BnP1 exerts biological effects similar to those of Jar on the hydrolysis of coagulation factors, the degradation of extracellular matrix components, and the apoptosis induction in endothelial cells. Nevertheless, Jar possesses strong hemorrhagic activity, while BnP1 presents weak hemorrhagic activity [21].

The inflammatory response starts with sequential and orchestrated phenomena in the vascular endothelium, resulting in the loss of vasomotor reactivity and cell recruitment. Endothelial dysfunction causes an imbalance in microcirculatory homeostasis, activating the immune response that triggers the production and release of inflammatory mediators, such as tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1) and interleukin-6 (IL-6) and the subsequent release of vasoactive substances, alteration of blood flow and increased vascular permeability and leukocyte migration to inflamed tissues after *Bothrops* envenomation [19,22].

In addition, the release of these chemotactic agents mediates a sequence of adhesive contacts between leukocytes and endothelial cells [23]. These adhesive contacts, as well as the exit of these cells from postcapillary venules, are mediated by the expression of adhesion molecules [24]. Leukocyte transmigration to tissue is composed of a complex series of events depending on the time and expression levels of various adhesion molecules through a precise mechanism with additive/cooperative interaction potential that is mediated by the opening of junctions of adjacent endothelial cells (via para-cellular) or through the cell body (transcellular) [25,26].

Cell adhesion molecules are glycoproteins expressed on the cell surface that mediate contact between two cells or between cells and the endothelium. Three families of adhesion molecules have been identified: (1) selectins, which are predominantly responsible for the initial contact of leukocytes with the endothelial vasculature in the leukocyte recruitment and "rolling" stages [27]; (2) integrins, which are composed of the α subunit, also known as CD11, and β subunit and mediate the firm adhesion of leukocytes to the endothelium, especially integrins of the β2 or CD18 family [28]; and (3) CAMs (cell adhesion molecules), proteins belonging to the immunoglobulin superfamily that are involved in adhesion and migration between leukocytes and endothelial cells [27,28].

Few studies have examined the participation of different domains of metalloproteases in inflammatory reactions, and even fewer have assessed their roles in modulating the expression of adhesion molecules during the process of leukocyte recruitment and cell migration after exposure to these toxins. An understanding of the mechanisms that contribute to the inflammatory response caused by *Bothrops* envenomation may help resolve the local reaction observed. Thus, this study investigated the effects of different SVMP domains on modulating the expression of adhesion molecules on leukocytes and the microvasculature of mice exposed to three different toxins isolated from *Bothrops* venoms: Jar and Jar-C, which are a PIII-SVMP and disintegrin-like protein isolated from *Bj*V, respectively, and BnP1, a PI-SVMP with weak hemorrhagic action isolated from *Bn*V.

#### **2. Results**
