Patients with FH exhibit increased rates of atherosclerosis and high risks of myocardial infarction at a young age. These outcomes are mainly related to lifelong exposure to high plasma cholesterol levels.
Recent studies support the view that patients with FH, despite long-term cholesterol-lowering treatments, exhibit enhanced inflammation.12 Indeed, hypercholesterolemia and inflammation may be considered as “two sides of the same coin”.13 In the present study, circulating SORBS2 levels were observed to be higher in FH patients than in control participants. In particular, we observed positive correlations between circulating SORBS2 levels and inflammatory factors (IL-1β) and
between circulating SORBS2 levels and lipid indexes (TC and LDL-C), indicating that SORBS2 may play an important role in inflammation in patients with familial hypercholesterolemia. Based on these observations, we treated THP1-derived macrophages with Ox-LDL to mimic hypercholesterolemia, and then investigated whether SORBS2 could regulate inflammatory process and the formation of macrophage-derived foam cells, and thus contribute to the progression of atherosclerosis (AS) in hypercholesterolemic populations.
SORBS2 is a key factor bridging inflammatory dysfunction and atherosclerotic coronary artery disease. SORBS2 is the archetypal member of a three-protein family that includes CAP (SORBS1) and vinexin (SORBS3).14 The important role played by SORBS2 in various diseases has previously been reported.2, 14 SORBS2 variants are also known to be associated with CHD.15
Moreover, SORBS2 in human heart tissue, where it is localized in the Z-bands of mature myofibrils, has been reported to regulate important processes in cardiomyocytes.3, 16 In the present study, we report that SORBS2 expression is a marker for the progression of AS. Thus, circulating SORBS2 levels are significantly higher in patients with FH, and patients with FH have higher levels of inflammasome-related cytokines. Wang et al. previously reported that SORBS2 can modulate the levels of IL-6, TNF-α, and IL-1β in sepsis-associated cardiac dysfunction.17 Here, we found that SORBS2 silencing inhibited the secretion of IL-1β and IL-18 into culture supernatant after Ox-LDL induction of macrophage activation. Moreover, SORBS2 silencing suppressed IL-1β and IL-18 levels in cell lysate. Hence, SORBS2 silencing inhibits inflammation in Ox-LDL-induced foam cells.
The NLRP3 inflammasome, the best characterized pattern recognition receptor in innate immune response, is comprised of a sensor (NLRP3 protein), an adaptor (ASC protein), and an effector (pro-caspase-1). Epidemiological studies provide indirect evidence that patients with AS display high aortic expression of NLRP3, and that NLRP3 expression levels are correlated with disease severity and clinical risk factors.18 After activation stimulation, NLRP3 protein interacts with apoptosis-associated speck-like protein containing CARD (ASC) and pro-caspase-1 to form the NLRP3 inflammasome. This ultimately results in cleavage and maturation of the potent pro-inflammatory cytokines IL-1β and interleukin 18 (IL-18) by mature caspase-1.19, 20 Mechanistically, activation of the NLRP3 inflammasome proceeds through the NF-κB pathway.
In the present study, we confirmed that Ox-LDL-induced macrophages significantly promoted the activation of NF-κB. After NF-κB activation, NF-κB is translocated into the nucleus, thus enhancing NLRP3 inflammasome complex formation and activation. SORBS2 silencing effectively abolished both NF-κB activation and NLRP3 inflammasome activation in macrophages. When the NLRP3 inflammasome becomes activated, the adaptor protein ASC assembles into a large protein complex named an ASC speck, and this is considered a typical sign of inflammasome activation. ASC specks assembled in response to inflammasome activation can be visualized under a microscope as micrometer-sized foci. Here, we investigated the assembly of ASC specks in vitro by immunofluorescence. We found that SORBS2 silencing blocked the assembly of ASC specks in Ox-LDL stimulated macrophages.
Following ASC speck assemblys, ASC nucleates precursor caspase-1, and the NLRP3 inflammasome then cleaves precursor caspase-1 into active caspase-1. Subsequently, activated caspase-1 processes IL-1β and IL-18 precursors into mature IL-1β and IL-18, augmenting the inflammatory response and associated impairments. Our studies reveal that SORBS2 silencing inhibited activation of the NLRP3/ caspase-1/ IL-1β pathway, ultimately blocking the inflammation process in AS, and thus potentially providing protection against hypercholesterolemia and atherosclerosis.21
Activation of the NLRP3 inflammasome is reported to require additional triggers such as reactive oxygen species (ROS). ROS are unstable and highly reactive molecules produced by reduction of oxygen mainly during mitochondrial oxidative phosphorylation.22 In endothelial cells, Ox-LDL treatment under high glucose conditions increases intracellular ROS production, and this subsequently activates the NLRP3 inflammasome.23 Our findings reveal that SORBS2 silencing inhibited Ox-LDL-induced ROS production. By decreasing the production of oxidative stress markers, SORBS2 silencing further inhibits activation of the NLRP3 inflammasome.
In atherosclerosis, formation of macrophage foam cells is considered to be the initial step in the pathological process.24 These cells are known to regulate Ox-LDL uptake and intracellular cholesterol trafficking. Ox-LDL has been reported to play a causative role in the genesis and progression of AS which acts via binding to scavenger receptors and accumulating in the cytoplasm. Our data reveal that foam cell formation was enhanced in THP-1 cells under Ox-LDL conditions, as demonstrated by Oil Red O staining. It was previously established that receptor-mediated cholesterol efflux and reverse cholesterol transplant are major mechanisms in the removal of cellular cholesterol, and CE hydrolysis is the rate-limiting step in these processes. In Ox-LDL treated THP-1 cells, increased cholesterol efflux (TC, FC, and CE) was observed to downregulate ABCG1 and PPARγ. These results are consistent with the Bekkering et al. study. 25 Because SORBS2 silencing significantly increased TC, FC, and CE efflux, and it can be concluded that SORBS2 plays a role in Ox-LDL-induced macrophage foam cell formation.
The ligand-inducible transcription factors PPARγ is highly expressed in macrophages, and is known to control macrophage inflammation, polarization, and lipid metabolism in atherosclerosis plaques.26 Previous studies have demonstrated a crucial role for PPARγ in the induction of ABCA1/ ABCG1 expression, and the prevention of foam cell formation and atherosclerosis progression.27 Jiang el al. reported that PPARγ ligands markedly reduce induction of ABCG1 expression and HDL-mediated cholesterol efflux.28 In the present study, We found that SORBS2 silencing augmented ABCG1 protein expression and cholesterol efflux to HDL. In contrast, SORBS2 silencing did not influence ABCA1 protein expression and thus does not affect cholesterol efflux to ApoA1. Therefore, it can be speculated that SORBS2 silencing regulates macrophage foam cell formation by promoting reverse cholesterol transport. Taken together, our results provide further evidence that SORBS2 can regulate lipid biosynthesis through transcriptional activation of lipogenic genes.
In summary, our study provides evidence that SORBS2 silencing regulates foam cell formation and relieves inflammation by blocking activation of both the NLRP3 inflammasome and NF-κB. In addition, SORBS2 silencing reduces ROS production. Through these mechanisms, SORBS2 silencing prevents cellular lipid accumulation and promotes cholesterol efflux via the PPARγ/ABCG1 pathway. Our findings shed new light on the therapeutic mechanisms of hypercholesterolemia and atherosclerosis.