Mitochondrial dysfunction and abnormal telomere/telomerase balance lead to a persistent oxidative stress in SLE, mainly involving circulating leukocytes and ECs (20). The oxidative process induces cell adhesion molecules (CAMs) expression (21), with consequent higher leucocyte-endothelial cell interactions and leucocytes’ transmigration to sites of inflammation (22). In addition, a significant association between higher anti-double stranded-DNA (anti-dsDNA) antibodies levels and higher levels of oxidative products was reported (23, 24). The excessive production of reactive oxygen and nitrogen species (ROS and RNS) leads to modifications of different cellular molecules, such as proteins, lipids, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), generating neo-antigens with a consequent production of autoantibodies, and uncontrolled lymphocytes’ activation (23, 25). In SLE, three main targets of oxidative stress have been identified: oxidized lipids, oxidized low-density lipoprotein (LDL) and proinflammatory high-density lipoprotein (HDL), all playing a crucial role in pathogenesis of SLE-related ED and atherosclerosis (26, 27).

Proinflammatory cytokines play a direct role in accelerating SLE atherosclerosis. In particular, all three classes of interferons (IFNs), namely IFN-I (IFN-α, IFN-β, IFN-δ, IFN-ε, IFN-κ, IFN-τ, IFN-ω, and IFN-ζ), IFN-II (IFN-γ), IFN-III (IFN-λ1, IFN-λ2, and IFN-λ3), participated in the process of atherosclerosis (19). IFN-α and IFN-γ promote lipoproteins’ oxidation (28, 29) and ED by accelerating ECs apoptosis and damaging endothelial progenitor cells (EPCs) (28, 30), one of the vascular repair mechanisms. On the other hand, IFN-γ increases vascular smooth muscle cells’ (VSMC) proliferation and migration (31), VSMC and macrophages apoptosis in atherosclerotic plaques, inducing plaque instability (32). The long-term activation of IFN-I system induces the expression of different chemokine pathways that recruit leukocytes into inflammatory sites promoting the dysfunction of ECs and EPCs (19).

Neutrophil Extracellular Traps (NETs), a unique type of neutrophils communication characterized by the extrusion of chromatin and other molecules, are considered a key factor in SLE atherosclerosis (33). NETs can enhance vascular leakage, endothelial-to-mesenchymal transition (34) and ECs death (35). Moreover, NETs enhance oxidation processes (36), secretion of IFN-α (37), interleukin (IL)-1β (38), and activate coagulation cascade (39).

Many autoantibodies can affect endothelial function, by promoting pathogenic molecules and inhibiting potential protective factors (40). Antiphospholipid antibodies (aPL), that are anticardiolipin antibodies (aCL) and anti-β2-glycoprotein I antibodies (anti-β2GPI), can contribute to accelerated atherosclerosis by inducing a proinflammatory endothelial phenotype through a direct interaction with ECs (41). Different authors described ECs activation by aPL via EC-derived extracellular vesicles through a toll like receptor (TLR) 4 and 7-dependent pathway, resulting in paracrine stimulation of neighboring unstimulated ECs (42–44). In addition, aPL can upregulate the tissue factor expression on ECs and monocytes, and promote endothelial leukocyte adhesion and pro-inflammatory cytokine secretion (41). Finally, aPL are considered an independent predictor of atherosclerotic plaque progression in SLE (45). Other autoantibodies have been described as contributors of accelerated atherosclerosis in SLE: anti-HDL-IgG that induce LDL to enter the ECs; anti-apolipoprotein A1 (ApoA1)-IgG that activating the transcriptional nuclear factor kappaB (NF-kB) favor the expression of inflammatory factors at endothelial level (46); anti-FXa-IgG can inhibit FX enzyme (47), modifying the of hemostasis/thrombosis equilibrium and promoting ED (48). Moreover, anti-C1q antibodies play a role in atherosclerosis by reducing C1q’s level and lowering their protective effects on endothelium (49, 50).

In general, the subset of CD28- (CD28null) T cells is char- acterized by pro-inflammatory properties and plays an active role in destabilization of the plaque itself, increasing endothelial oxidative markers, and arterial stiffness (51). In humans, high levels of CD4+CD28null T cells, responsible of an aberrant T-B lymphocytes’ interaction, have been described during instable angina, and could be involved in the atherosclerotic plaque instability (52). The prevalence of these cells is increased in systemic autoimmune diseases because of the repeated antigenic stimulation that induces a downregulation of CD28 from the lymphocytes’ membrane (53). The so-called angiogenic T cells (Tang) are characterized by the expression of CD3, the platelet-endothelial cell adhesion molecule-1 (CD31) and the receptor for stromal cell factor-1 CXCR4 (54). Due to their ability to enhance endothelial repair function (55) and promote new vessel formation (54), Tang could be used as a novel putative biological marker for CVD. A higher number of circulating Tang may be involved in ED among several autoimmune diseases, such as rheumatoid arthritis (RA), SLE and SSc, as a consequence of endothelial damage or an inefficient angiogenesis (56–58). Accordingly, in a recent study of our group, we demonstrated that the nail video-capillaroscopy (NVC) alterations in a cohort of patients with SLE and without traditional CV risk factors were associated with ED and with the increase of circulating Tang (59). A subtype of Tang called “aging” Tang (CD28null-Tang) seems to be not protective but cytotoxic, due to their ability to secrete inflammatory mediators and release cytolytic molecules from intracellular particles, inducing EC damage and accelerated atherosclerosis in most SLE patients (60). Moreover, CD28null-Tang increased in SLE patients with traditional CV risk factors and active disease (60). In our recent experience, we observed that the rate of circulating pro-angiogenic Tang decreased very early in disease course, with an increase of the rate of the “aging” CD28null subset. Our preliminary data suggest that Tang might exert their effects on the endothelium via the pro-angiogenic mediators IL-8 and metalloproteinase-9 (61). Another T lymphocyte subtype, regulatory T cells (Treg), are believed to play a protective role in autoimmune diseases. Anyway, atherosclerosis’s severity does not seem to be strictly related to their numbers, but rather to their dysfunction (62, 63). In SLE, Treg cells are significantly reduced in both, number and function (64). In human studies and mouse models, Treg have been associated with a protective role in atherosclerosis (65) and their decrease is significantly associated with acute coronary events (18). Recently, the invariant natural killer T cells showed an anti-atherosclerotic phenotype in SLE patients and can induces macrophages to polarize into anti-inflammatory and anti-atherosclerotic M2 phenotype (66).

Endothelial progenitor cells are a group of bone marrow-derived cells, acting in vascular homeostasis control and endothelial repair (67). Some authors reported a reduced number of EPCs in patients with CV risk factors (68) and CVD (69). Therefore, EPCs could be considered a new marker of CV risk, especially in SLE patients in which traditional CV prediction models fail to estimate the risk of clinical CVD. Physiologically, after endothelial injury, vascular repair occurs by accelerating the replacement of ECs: a process that involves proliferation and migration of adjacent ECs and resident EPCs and recruitment of new EPCs. Although data in SLE are controversial, EPCs are reduced in number and are functionally impaired (19). This impairment seems to be the result of the balance between risk factors (including IFN-I) and protective factors (including Tang cells). In particular, IFN-I accelerates SLE atherosclerosis, by interfering with EPCs (19), as suggested by studies in adult- or childhood-onset SLE (67, 70). The results among studies are difficult to be compared because EPCs could be identified using different and not yet standardized methods, such as flow cytometry or through different cell isolation techniques (67). Type I IFN, overexpressed during a SLE flare and involved in SLE pathogenesis, was described as a contributor of EPCs dysfunction in the disease (67). Furthermore, some data demonstrated that recombinant IFN-α displays a toxic effect on CD133/CD34 + cells (e.g., putative EPCs) in culture. The use of monoclonal antibody blocking IFN pathways in SLE leads to a normalization of EPCs function (71).

Repetitive ischemia and reperfusion processes causes oxidative stress with subsequent tissue damage in SSc, mediated by proinflammatory cytokines and activated leukocytes. These activated leukocytes also show increased expression of inducible nitric oxide synthase (iNOS), leading to the production of a huge amount of NO that reacts with oxygen in the re-perfused blood to form ROS. This causes a direct endothelial injury that leads to vasoconstriction and conversion to a procoagulant phenotype (86).

The dysregulation of EC within the vascular wall has a major role in the above-mentioned fibroproliferative vasculopathy (87). This contribute to the unbalanced production of vasoactive mediators resulting in vasoconstriction (88, 89). The alterations of mediators involved in this process were described as both quantitative and qualitative. A particular mention has to be done with regards to the alterations of the vascular endothelial growth factor (VEGF). In fact, despite the fact that higher circulating levels of this vasodilator agent were described in SSc patients in comparison with healthy controls, anti-angiogenic VEGF isoform was strongly expressed in the skin of SSc patients (90). In addition, the increased expression of adhesion molecules by damaged endothelial surface promotes leukocyte trans-endothelial migration, activation, and accumulation (91, 92). ECs transdifferentiate into myofibroblasts gaining mesenchymal cell markers (93, 94). These events culminate in the intima-media proliferation and vessel occlusion leading to tissue hypoxia, which further promotes cell injury and fibroblasts activation (87). Viral infections, coagulation cascade activation, complement system impairment and antibodies against ECs have been proposed as the initial trigger in SSc pathogenesis (95, 96). Some viral infections have been linked to activation/injury of ECs through a mechanism of molecular mimicry. For instance, human cytomegalovirus infection induces antibodies that recognize an amino acid sequence on a viral protein, which is homologous to a surface molecule highly expressed on ECs, inducing apoptosis of ECs (97). Some studies have found a correlation between the parvovirus B19 DNA expression levels and the severity of ED in SSc (98, 99). Recently, new evidence focuses on whether SARS-CoV-2 infection triggers autoimmunity and may have a role in SSc pathogenesis. Indeed, exploration of the SARS-CoV-2-related endotheliitis might provide further important information in the understanding of the early SSc pathogenesis (100).

The complement system role in the pathogenesis of SSc vasculopathy has not been exhaustively studied. Its classical functions such as opsonization, recruitment of inflammatory cells, influence of coagulation cascade and angiogenesis are primary for ECs integrity. In normal conditions, complement attack is tightly regulated by regulatory proteins, ensuring protection of EC layer. A reduced expression of these regulators has been shown in SSc skin, potentially leading to endothelium-bound membrane attack complex of complement deposition that could cause EC apoptosis (101).

The anti-endothelial cell antibodies (AECAs) can be found in almost 50% of SSc patients and can react with various cell surface antigens on ECs leading them to apoptosis (102) through the antibody-dependent cell-mediated cytotoxicity mechanism (103–105). An association between circulating antibodies and vascular manifestations has been described for antibodies against cell surface receptors such as angiotensin II type 1 receptor and endothelin-1 type A receptor (106). Among other antibodies possibly associated with vasculopathy in SSc, aPL should be considered. Their frequency in SSc is highly heterogeneous and ranges from 0 to 57% (107). Sobanski et al., carried out a meta-analysis, revealing an overall pooled prevalence of 14% (108). ACL and anti-β2-GPI antibodies can contribute to accelerated atherosclerosis by interacting with ECs and inducing a proinflammatory endothelial phenotype (41). Some studies reported an association between aPL positivity and PAH and DU (109–113), while others did not (108, 114, 115). Lastly, considering the strong clinical associations of SSc specific antibodies (anticentromere, anti-topoisomerase 1, anti-RNA polymerase III and anti-Th/To antibodies) and their role as prognostic biomarkers, a potential pathogenicity of these antibodies was suggested. Raschi et al., demonstrated that SSc specific antibodies bound to their antigens to form immune complexes (ICs) elicit pro-inflammatory and pro-fibrotic effects on healthy ECs (95). They stated that immune complexes composed with SSc specific antibodies might contribute to scleroderma pathogenesis through a direct interaction with TLRs. Endothelial incubation with SSc-ICs modulates several molecules (endothelin-1, IL-8, inter-CAM-1, IL-6, and transforming growth factor β1) involved in the three cardinal scleroderma pathophysiologic processes (95).

As previously outlined, Tang are required for endothelial progenitor colony formation, promote new vessel formation by secreting angiogenic factors such as VEGF and adhere to ECs. Tang can interact directly with the CD31 expressed by ECs via endothelial-T-cell CD31-CD31 homophilic interactions. In addition, given that these cells also express the cytotoxins granzyme B and perforin, they also have cytotoxic potential. Zhang et al., reported that these cells secrete large amounts of proinflammatory cytokines, such as tumor necrosis factor alpha, IL-6 and IFN-γ, confirming their proinflammatory features (116). Interactions these Tang related cytokines may contribute to ED by exacerbating oxidative stress and reducing phosphorylation of endothelial NOS (117). Their frequency is increased in individuals with traditional CV risk factors further supporting their role in regulating ED (60). It was found that circulating Tang were significantly increased in SSc patients with DU compared either with SSc patients without DU or with healthy controls. In addition, in SSc patients, Tang levels correlate with NVC patterns: higher levels were observed in patients presenting late NVC pattern more frequently than in those with early/active NVC patterns (58). In another study, the absolute number of Tang was higher in SSc patients compared to healthy controls, especially in SSc patients with PAH (118). Taken together, these findings demonstrated that Tang are expanded in SSc patients displaying severe peripheral vascular complications suggesting that circulating Tang increase as a reaction to ischemia and might represent a novel biomarker closely reflecting the severity of SSc-related peripheral vasculopathy.

The scleroderma impairment of neovascularization could be associated to both angiogenesis and vasculogenesis failure. Besides insufficient angiogenesis, the contribution of defective vasculogenesis to SSc vasculopathy has been extensively studied (119). As mentioned above, EPCs are defined as circulating primitive cells that contribute to postnatal vasculogenesis (120) and, in SSc patients, circulating EPCs were shown to be reduced in comparison with healthy controls (121). In addition to quantitative alterations, an impaired potential of SSc-derived EPCs to differentiate into mature ECs was reported in terms of functional properties of EPCs (122). It was suggested that EPC precursors were functionally altered before their release into the bloodstream because of a dysregulated microenvironment within the bone marrow (reduced microvascular density and increased fibrosis) (123, 124). In addition, the hypoxic condition of the affected tissues of SSc patients are known to stimulate the differentiation of monocytic EPCs, one EPCs subset (125), through activation of hypoxia-inducible factor (HIF)-1α (126). These local stimuli promote the accumulation of functionally altered monocytic EPCs into the affected lesions of SSc and, since monocytic EPCs are capable of differentiating into cells that produce extracellular matrix proteins (127, 128), they might participate in the fibrotic process in the affected organs (128, 129).