**1. Introduction**

Cardiovascular diseases (CVD) are a group of pathologies of the vascular system that are growing in number and that usually present a build-up of fatty deposits inside the arteries (atherosclerosis) and an increased risk of blood clots and can also be associated with damage to arteries in organs such as the brain, heart, kidneys, and eyes. CVD are triggered by several risk factors, ranging from pathologies such as diabetes, hypercholesterolemia, and metabolic syndrome, to unhealthy habits, e.g., inactivity, smoking, and excessive alcohol. Apart from the medical advice that may include changing lifestyle and diet, the only medical treatment that can help decrease the risk is lowering cholesterol levels and controlling blood pressure and coagulation [1,2]. Despite the high numbers of data about CVD, the exact timing and molecular mechanism at the basis of the pathologies are still unclear, even though they are all inflammation related [3,4]. Some authors proposed that some of the risk factors lead to qualitative changes in the endothelium such as changes in permeability and increase in adhesion molecules expression that attract leucocytes ("response of injury" hypothesis) [5,6], which, in turn, cause the inflammatory condition. Moreover, the localized inflammation causes the thickening of the arterial wall due to the increased deposition of extracellular matrix (ECM) and the newly formed ECM traps lipoprotein and inflammatory/growth factors from the circulation within the vessel wall [3].

In obese patients, the endothelium is characterized by dysfunction associated with a condition of vascular low-grade inflammation in which an excess of TNF-α (tumor necrosis factor) is generated either in small vessels or within the perivascular adipose tissue [2]. TNF-α has also an effect on the release of nitric oxide (NO), which is produced to regulate vascular tone, cardiac contractility, and vascular remodeling [7].

**Citation:** Caravà, E.; Moretto, P.; Caon, I.; Parnigoni, A.; Passi, A.; Karousou, E.; Vigetti, D.; Canino, J.; Canobbio, I.; Viola, M. HA and HS Changes in Endothelial Inflammatory Activation. *Biomolecules* **2021**, *11*, 809. https://doi.org/10.3390/ biom11060809

Academic Editors: Dragana Nikitovic and Serge Perez

Received: 8 February 2021 Accepted: 25 May 2021 Published: 29 May 2021

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The endothelial layer is composed of several components, the major ones including fibrous proteins such as collagen, fibronectin, elastin, laminin, and glycosaminoglycans (GAGs) chains such as hyaluronan (HA) or proteoglycans (PGs). The PGs and GAGs fill the interstitial space of tissues left free from fibrous protein, forming a well-organized network conferring tissue hydration [8] as well as function as co-receptors and therefore regulating their activity and that of growth factors and cytokines [1,9]. Both HA and heparan sulfate (HS) GAGs are pivotal in the maintenance of endothelial function, and their removal causes a loss of barrier properties comparable to an inflammatory condition [10]; moreover, several data pointed out the Syndecan family of HSPG as central in the endothelium behavior [11]. Syndecans are a family of four transmembrane HSPGs that are expressed in a cell-typespecific manner [11]; Syndecan-1 is present during development and in adult endothelium and cancer cells; Syndecan-2 is expressed in liver, mesenchymal tissue, and neuronal cells; Syndecan-3 is a neuronal type; and Syndecan-4 is ubiquitously distributed [12,13]. The complex mechanism of biosynthesis and modification of the HS chains generate a variability in N-sulfation levels along the polysaccharides. EXT1 and EXT2 are the polymerizing enzymes, while glucosaminyl N-deacetylase/N-sulfotransferase (NDST) is the first modification enzyme that starts to work on the growing heparan sulfate (HS) polysaccharide chain. This enzyme defines the sulphation pattern, which will determine the ability of the HS chain to interact with target molecules [14]. Moreover, the NDST1 has the capacity to bind to EXT2, and EXT1 and EXT2 expressions affect the N-sulfation degree, hinting that the overexpression of all the three enzymes happens simultaneously. The subsequent sulfations occur on the growing chains [15]; it is noteworthy that although there is only a single 2-O-sulfotransferase, there are three 6-O-sulfotransferases (6OST1- 3) and seven 3-O-sulfotransferases [16], indicating the great importance of the sulfation pattern on the growth factor binding ability of the HS chains [17]. It produces regions rich in N-acetylated residue (GlcA and GlcNAc) called NA domain, regions rich in Nsulfated residue (IdoA and GlcNS derivates) called NS domain, and sequences that contain alternation of NA and NS. The ligands binding mainly depend upon the distribution of these domains [18,19]. The synthetic GAG machinery can be altered by several events, including the changes in PG core proteins expression or UDP-sugars transporters [20].

A preliminary clinical study in patients with resistant arterial hypertension (CVD risk factor) indicates high Syndecan-4 level as a potential marker for endothelial dysfunction [21]; moreover, the same HSPG has also been shown to regulate focal adhesions junctions, demonstrating the properties of a mechano-transducer effector [22], and importantly, its fragments generated following thrombin cleavage can modulate changes in endothelial barrier resistance [23]. Since one of the major events in atherosclerosis onset is the accumulation in the sub-endothelium of lipids driven by the lipoprotein LDL, the passage of such particles through the endothelial barrier is a critical step. Nevertheless, despite the many studies underscoring the relevance of plasma lipoproteins and the effects of lipids and cholesterol on the cell behavior and extracellular matrix architecture of the tunica intima [3], the data about the events causing the LDL transcytosis and accumulations are still scant. Links regarding Syndecan-4 metabolism/levels and the endothelial barrier system are not yet known.

Following the data reported, we made endothelial cells undergo inflammatory conditions using the TNF-α cytokine and investigated the effect in the main ECM components, i.e., HA and Syndecans, evaluating their effect on cell behavior, with regards to the proatherogenic aspect of membrane permeability.
