oxLDL impact caveolae/Cav -1 signaling
In EC, caveolae sense and transduce hemodynamic changes into biochemical signals to regulate vascular function. Caveolae compartmentalize signaling proteins in the plasma membrane through direct/indirect interactions with Cav-1. These interactions allow to fine-tune the magnitude of signaling cascades.
Within caveolae, Cav-1 functions as scaffolds for several proteins such as eNOS (Shaul, 2003; Williams and Lisanti, 2004) and NADPH oxidase (Patel and Insel, 2009; Chen et al ., 2014).
NO is a potent vasodilator and anti-inflammatory mediator produced by a family of enzymes called NOS. Three NOS isoforms have been identified: neuronal NOS (nNOS; NOS1), inducible NOS (iNOS, NOS2) and endothelial NOS (eNOS; NOS3) all of which differ slightly in function and structure. NO is synthesised in vascular EC from the abundant amino acid L-arginine by eNOS. This enzyme is constitutively expressed in EC where its product acts as vasoprotective molecule able to regulate the vascular tone and to attenuate both platelet aggregation and neutrophil-endothelium interaction (Brunner et al ., 2003). eNOS has a slow basal activity for NO generation that in EC is enhanced by agonists such as acetylcholine, bradykinin and histamine, which increase intracellular calcium, whereas shear stress and hormones increase eNOS activity independently of changes in intracellular calcium (Chen et al ., 2018).
The eNOS isoform is abundantly represented in EC of the vascular intima and it is mainly located in the plasma membrane caveolae where it can also be found also associated with the protein Cav-1. It is important to note that eNOS is also found in the Golgi apparatus, cytosol, cytoskeleton, and even in the nucleus. However, Fulton and coworkers (2002) demonstrated that eNOS is principally active on the plasma membrane.
The interaction between eNOS and Cav-1 has been observed both in vivo and in vitro . Through colocalization and coimmunoprecipitation experiments it has been shown that binding of eNOS to Cav-1 inhibits the enzyme activity, resulting in reduced NO production (Bucci et al ., 2000).
In this context, it has been demonstrated that oxLDL cause selective depletion of cholesterol within the caveolae (Shaul, 2003). This event results in eNOS intracellular redistribution and an attenuated capacity to activate the enzyme (Figure 2). In addition, oxLDL promote the expression of several pro-inflammatory mediators, including iNOS, presumably via the MAPKs/NF-kB pathway. This leads to an unbalance between eNOS and iNOS activity with the production of high amount of NO that works as free radical with bactericidal and inflammatory function (Figure 2) (Gliozzi et al . 2019). The production of high NO concentrations by iNOS causes the generation of high levels of peroxynitrite which has been correlated with apoptosis of EC (Salveminiet al ., 2013). Gliozzi and coworkers suggested that this inflammatory condition promotes the nuclear translocation of NF-kB failing the protective mechanisms as autophagy (Gliozzi et al . 2019; Mollace et al ., 2015). These findings are in line with our previously reported data (Luchetti et al ., 2019) showing that high concentrations of secosterol B promotes cell apoptosis via a pathway that involves early phosphorylation of eIF2α and NF-kB activation, suggesting that the adaptive program fails, and the cell activates the apoptotic program. Recently, Potje et al . (2019) demonstrated that cholesterol depletion affects the number of caveolae promoting eNOS uncoupling that results in Nox-dependent O2 production at the expense of NO generation.
One interesting finding is that Cav-1 phosphorylation at Tyr(14), following LPS exposure, favors Cav-1 and Toll-like receptor 4 (TLR4) interaction and, thereby, TLR4 activation of MyD88, leading to NF-κB activation and generation of proinflammatory cytokines (Jiao et al ., 2013). Notably, the effects observed in LPS-treated cells are mimicked by high-mobility group box 1 (HMGB1), a protein known to accumulate in atherosclerotic lesions and to mediate vascular inflammation. TLR4 activation by HMGB1 in HAEC cells has been demonstrated by Yang et al. (2016), as evidenced by expression of its downstream partner MyD88. Treatment with recombinant HMGB1 increased ERK phosphorylation and nuclear translocation of NF-κB. Thus, HMGB1-induced activation of TLRs initiates pro-inflammatory signaling pathways and mediates the release of cytokines and chemokines, thus contributing to vascular inflammation and endothelial dysfunction (Jiao et al., 2013) (Figure 2). While there is evidence that oxLDL can promote cytoplasmic relocation and extracellular release of HMGB1 by EC (Zhouet al ., 2016; Yu et al ., 2012), the role of caveolin in HMGB1-induced TLR4 activation is not clear. Notably, HMGB1 increases endothelial cell Cav-1 and TLR4 protein expression, suggesting that TLR4 and Cav-1 may act together. These proteins colocalize in HUVEC cells and knockdown of TLR4 abrogates Cav-1 induction (Jiang et al ., 2014). More recently, Lin et al. (2018), provided evidence that oxLDL promote phosphorylation of Cav-1 in HUVEC and increase oxLDL uptake. Intracellular accumulation of oxLDL induced NF-κB activation and HMGB1 translocation from nucleus to cytoplasm resulting in cell apoptosis. NF-κB activation also facilitated Cav-1 phosphorylation and HMGB1 expression. Considering that HMGB1 enhances oxLDL uptake through induction of LOX-1 (Lee et al ., 2012), it is plausible that a tight crosstalk between HMGB1, TLR4, NF-kB, LOX-1 and caveolin may occur in response to oxLDL.
Only few studies concerning the impact of oxLDL/caveolae interaction on caveolae/Cav-1-mediated signalling are reported in the literature. Therefore, more research focusing on caveolae/Cav-1 and oxLDL signal transduction is urgent in order to better understand the mechanism of atheroma formation.