Macrophage P2Y12 regulates iron transport and its inhibition protects against atherosclerosis.

Iron retention is commonly observed in atherosclerotic plaques and is believed to be detrimental to atherosclerosis. Platelet P2Y12 is a target of antiplatelet therapy in preventing thrombotic complications of atherosclerosis. The protective effect of P2Y12 on hematopoiesis reported by our previous work implies the involvement of P2Y12 in iron metabolism. This study further investigated the role of P2Y12 in the iron metabolism of macrophages, the key player in systemic iron homeostasis and atherosclerosis. The association between serum iron and the use of P2Y12 inhibitors was evaluated by a case-control study in human. Secondary iron overload and atherosclerosis animal models were established in P2Y12-deficient zebrafish to explore the role of P2Y12 in macrophage iron metabolism in vivo. Both iron-overloaded murine primary peritoneal macrophages (PMs) and ox-LDL-treated PMs with P2Y12 knockdown were used for in vitro studies. RNA sequencing and pharmacological approaches were performed to investigate the downstream mechanisms. Increased serum iron level was positively associated with P2Y12 inhibitor usage [odds ratio (OR) = 10.333 (1.281-83.370)]. Elevated serum iron level and transferrin saturation, reduced hepatic and splenic iron content, and decreased iron staining in macrophages were observed in secondary iron overload P2Y12-deficient zebrafish. Deficiency of P2Y12 in ApoEb-/- zebrafish fed a high-fat diet reduced atherosclerosis progression and intraplaque iron retention. Furthermore, reduced ferritin, restored cell viability and expression of ferroptosis marker proteins, and decreased ROS formation and inflammatory cytokines were observed in both iron-overloaded and ox-LDL-treated PMs with P2Y12 knockdown in vitro, while reversed phenotypes were observed after agonist-induced P2Y12 activation. Mechanistically, P2Y12 inhibition in iron-overloaded or ox-LDL-treated PMs suppressed NF-κB p65 phosphorylation and hepcidin expression, both of which were reversed by P2Y12 activation. P2Y12 inhibition decreased hepcidin autocrine through repressing NF-κB p65 phosphorylation in macrophages, preventing intracellular iron retention and atherosclerosis.

Hu YX ，You HM ，Bai MR ，Yue WH ，Li FF ，Hu BW ，Chen YS ，Shen XY ，Wu Y ，Wang JM ，He ZQ ，Tao X ，Jing Q ，Liang C ... -  《-》

Macrophage iron retention aggravates atherosclerosis: Evidence for the role of autocrine formation of hepcidin in plaque macrophages.

Iron accumulation has been frequently found in atherosclerotic lesions, especially in macrophages/foam cells, but the exact mechanisms by which hepcidin induces iron retention in plaque macrophages and its roles in atherogenesis remain unknown. Double immunofluorescence staining showed colocalization of hepcidin-positive macrophages with ox-LDL, TLR4, p-p65 and ferritin light chain (ferritin-L) both in human and murine atherosclerotic lesions. RAW264.7 macrophages incubated with ox-LDL showed elevated expression of TLR4, p-p65, hepcidin, ferritin-L/H, CYP27A1, CD36, PPARγ, liver X receptor α (LXRα), and ATP binding cassette transporter A1/G1 (ABCA1/G1), as well as increased intracellular labile iron pool level and lipid accumulation. Ox-LDL-induced iron retention and lipid accumulation were aggravated by lipopolysaccharide but blocked by TAK-242, an antagonist of TLR4. Moreover, macrophage TLR4/NF-κB pathway activation and foaming triggered by ox-LDL was enhanced by ferric ammonium citrate or exogenous hepcidin but attenuated by hepcidin silencing or the use of iron chelator. Meanwhile, the addition of hepcidin stimulated CD36-mediated Dil-labeled-ox-LDL uptake and inhibited the LXRα-ABCA1/G1 pathway-dependent cholesterol efflux in macrophages, which was significantly reversed by 27-hydroxycholesterol but further exacerbated by cyclosporin A, a selective inhibitor of CYP27A1. Our study provided the evidence that iron trapped in atherosclerosis plaque macrophages contributes to cholesterol disequilibrium-initiated foam cell formation, which is provoked by the unique but largely unknown autocrine formation of hepcidin in plaque macrophages via activating the TLR4/NF-κB pathway when exposed to ox-LDL. Such findings, considering the intricate vicious cycle between macrophage hepcidin autocrine-triggered iron retention and cholesterol disequilibrium, may shed new light on the "iron hypothesis" of atherosclerosis.

Xiao L ，Luo G ，Guo X ，Jiang C ，Zeng H ，Zhou F ，Li Y ，Yu J ，Yao P ... -  《-》

Hepcidin Deficiency Protects Against Atherosclerosis.

Malhotra R ，Wunderer F ，Barnes HJ ，Bagchi A ，Buswell MD ，O'Rourke CD ，Slocum CL ，Ledsky CD ，Peneyra KM ，Sigurslid H ，Corman B ，Johansson KB ，Rhee DK ，Bloch KD ，Bloch DB ... -  《-》

Disocin prevents postmenopausal atherosclerosis in ovariectomized LDLR-/- mice through a PGC-1α/ERα pathway leading to promotion of autophagy and inhibition of oxidative stress, inflammation and apoptosis.

Atherosclerosis (AS) is one of the major causes leading to mortality of dysfunctional cardiovascular events in the menopausal women, which has long-term deficiency of estrogen. At present, the primary treatment for postmenopausal AS is hormone replacement therapy (HRT). However, it can increase the risks of ovarian and uterine cancers with long-term therapy. So seeking for a phytoestrogen which can overcome the disadvantages of HRT is a great mission. Dioscin, a traditional Chinese medicine, extracted from the roots of dioscorea nipponica, has anti-inflammatory, anti-tumor and anti-apoptosis activities. Especially, it also has estrogenic activity. Thus, this study aims to investigate the effects of dioscin on postmenopausal AS. Currently, ovariectomy (OVX) is the accepted model for AS associated with estrogen deficiency, and it can mimic the cessation of ovarian function that occurs in postmenopausal women as well. We used the high fat diet and ovariectomy(HFD-OVX)model to induce postmenopausal AS in the low-density lipoprotein receptor- deficient (LDLR-/-) mice. (1) The levels of TG, TC, LDL-C, HDLC, MDA, GSH, MDA and GSH in serum of HFD-OVX induced LDLR-/- mice were measured by colorimetric assay. (2) The artery injury of HFD-OVX induced LDLR-/- mice was detected with Oil Red O staining. (3) The protein expressions of NOX4, P22phox, IκB, p-p65, n-p65, ICAM-1, VCAM-1, caspase-3, caspase-9, bcl-2, PGC-1α, ERα, ERβ in the arterial tissue of HFD-OVX induced LDLR-/- mice were detected by Western blot analysis. In vitro, the model of human aortic endothelial cells (HAECs) induced by oxidized low-density lipoprotein (ox-LDL) (150 μg /ml) was established, and the molecular mechanism of dioscin on atherosclerosis in postmenopausal women was investigated. (1) The levels of MDA, GSH, MDA and GSH in ox-LDL induced HAECs were measured by colorimetric assay. (2) Reactive Oxygen Species (ROS) of ox-LDL induced HAEC cells was detected by fluorescence staining. (3) The protein expressions of PGC-1α, ERα, ERβ, NOX4, P22phox, IκB, p-p65, n-p65, ICAM-1, VCAM-1, caspase-3, caspase-9, bcl-2 and LC3 in ox-LDL induced HAECs were detected by Western blot analysis. (4) The autophagy level of ox-LDL induced HAECs was measured by transmission electron microscopy. (5) The applications of si-RNA transfection were used to explore whether dioscin could activate PGC-1α/ERα pathway to inhibit postmenopausal atherosclerosis. In vivo, we found that dioscin decreased the level of TG, TC, LDL-C and increased the level of HDLC in serum of HFD-OVX induced LDLR-/- mice, and it has protective effects to maintain the lipid homeostasis; The Oil Red O staining study showed that dioscin could significantly inhibit the formation of atherosclerotic plaques in HFD-OVX-treated LDLR-/- mice; Dioscin decreased the levels of NOX4, P22phox, p-p65, n-p65, ICAM-1, VCAM-1, caspase-3, caspase-9, but increased the levels of HDL-C, GSH, SOD, PGC-1α, ERα, ERβ, IκB, Bcl-2 and elevated the autophagy level in arterial tissues of HFD-OVX induced LDLR-/- mice. It is particularly worth mentioning that the up-regulating effect of dioscin on ERα is stronger than ERβ in OVX treated mice. In vitro, the results of colorimetric assay showed that dioscin decreased the level of MDA and LDH, increased the level of SOD and GSH in ox-LDL-induced HAEC cells; Dioscin also suppressed the release of ROS in ox-LDL-induced HAECs by fluorescence staining; Dioscin decreased the levels of NOX4, P22phox, p-p65, n-p65, ICAM-1, VCAM-1, caspase-3, caspase-9, but increased the levels of PGC-1α, ERα, ERβ, IκB, Bcl-2 and the ratio of LC3-II/LC3-I in ox-LDL-induced HAECs; Dioscin significantly elevated the autophagy level of ox-LDL-induced HAECs by transmission electron microscopy analysis; In addition, by si-RNA transfection, we found that the inhibitory effects of dioscin on oxidative stress, inflammatory response and apoptosis might partly through PGC-1α/ERα pathway in ox-LDL induced HAECs. The data of dual-Luciferase reporter assay revealed that dioscin activated ERα at least partly through PGC-1α pathway. Dioscin significantly inhibited oxidative stress, inflammatory response, apoptosis and increased the level of autophagy in vivo and vitro. In addition, dioscin could regulate the balance of lipid metabolism. Moreover, we proved that the effects of dioscin attenuating postmenopausal atherosclerosis by inhibiting oxidative stress, inflammation and apoptosis were partly dependent on PGC-1α/ERα pathway. Therefore, dioscin, as a phytoestrogen, might become a drug for the treatment of atherosclerosis in postmenopausal women.

Yang Q ，Wang C ，Jin Y ，Ma X ，Xie T ，Wang J ，Liu K ，Sun H ... -  《-》

Moxibustion's protective role against atherosclerosis: Inhibition of Ca(2+) overload-triggered oxidative stress and inflammatory response via P2Y12/PI3K/AKT pathway.

This study aims to investigate the protective mechanism of moxibustion in combating atherosclerosis (AS). Apolipoprotein E (ApoE)-deficient mice, aged 8 weeks, were randomly assigned into four groups: the model group (n = 6), SC79 group (n = 6), moxibustion group (n = 6), and moxibustion+SC79 group (n = 6). All mice were fed with a high-fat diet (HFD). Concurrently, 8-week-old C57BL/6 mice of the same genetic background were utilized as the control group (n = 6) and were given a regular diet. Macrophages were isolated via flow cytometry. The intracellular Ca2+ expression in macrophages was evaluated, and aortic plaques were quantitatively assessed through en face oil red O and Masson staining. The presence of macrophages and smooth muscle cells in AS plaques was determined by MAC-3 and α-smooth muscle actin (α-SMA) immunohistochemistry. The relative fluorescence intensity of nuclear factor-κB (NF-κB) in macrophages was identified by immunofluorescence staining. The expressions of proteins related to the P2Y12/phosphatidylinositol 3-hydroxy kinase (PI3K)/protein kinase B (AKT) signaling pathway were examined by Western blotting. Moxibustion reduced free Ca2+ expression in macrophage cytoplasm, inhibiting Ca2+ influx and oxidative stress. Significant reductions in atherosclerotic plaque formation and inflammation markers, including TNF-α and IL-1β, were noted in the moxibustion group. Moxibustion modulated the P2Y12/PI3K/AKT pathway, impacting various inflammatory and oxidative stress-related proteins. Introduction of the AKT activator SC79 counteracted moxibustion's benefits, highlighting the P2Y12/PI3K/AKT pathway's central role. Moxibustion, through the P2Y12/PI3K/AKT signaling pathway, can inhibit Ca2+ overload-induced oxidative stress and inflammatory response, decrease macrophage infiltration, and increase the content of smooth muscle cells and collagen, thereby exerting a protective effect against AS.

Yang J ，Liu S ，Hu S ，Dai H ，Cai K ... -  《-》