Homocysteine promotes atherosclerosis through macrophage pyroptosis via endoplasmic reticulum stress and calcium disorder

Abstract

Background: Elevated plasma homocysteine levels, known as hyperhomocysteinemia, have been identified as an independent risk factor for atherosclerosis and related cardiovascular diseases. Macrophage pyroptosis-mediated inflammation is crucial in the development of atherosclerosis, but the underlying mechanisms remain unclear.

Methods: A hyperhomocysteinemia atherosclerotic model with ApoE^-/- mice fed with a high-methionine diet was constructed to investigate the role of plasma homocysteine in atherosclerosis. THP-1-derived macrophages were used to investigate the mechanisms by which Hcy regulates pyroptosis.

Results: We found that hyperhomocysteinemia resulted in larger atherosclerotic plaques and more secretion of inflammatory cytokines, while these effects were attenuated in Caspase-1 knockdown mice. Likewise, in vitro experiments demonstrated that treatment of macrophages with homocysteine resulted in NLRP3 inflammasome activation and pyroptosis, as evidenced by cleavage of Caspase-1, production of downstream IL-1β, elevation of lactate dehydrogenase activity, and extensive propidium iodide-positive staining of cells. These were all inhibited by Caspase-1 inhibitor. In addition, excessive generation of reactive oxygen species was associated with mitochondrial dysfunction, characterized by loss of mitochondrial membrane potential and ATP synthesis. Moreover, further experiments revealed that homocysteine induced endoplasmic reticulum stress, enhanced communication between the endoplasmic reticulum and mitochondria, and consequently contributed to calcium disorder. Furthermore, the endoplasmic reticulum stress inhibitor, 4PBA, the calcium chelator, BAPTA, and calcium channel inhibitor, 2-APB significantly improved macrophage pyroptosis.

Conclusion: Homocysteine accelerates atherosclerosis progression by enhancing macrophages pyroptosis via promoting endoplasmic reticulum stress, endoplasmic reticulum-mitochondria coupling, and disturbing of calcium disorder.

Keywords: Atherosclerosis; Calcium disorder; Homocysteine; Hyperhomocysteinemia; Inflammasome; Pyroptosis; Risk factor.

Fig. 1

HHcy accelerates the progression of atherosclerosis in ApoE^−/− mice. A Plasma levels of Hcy assessed by ELISA. B The images of whole aorta and aortic arch. The scale bars correspond to 1 mm. C Representative images of Oil-red O staining of cardiac aorta in mice of two groups (n = 5). The scale bars correspond to 1 mm. D Oil-red O staining was performed on frozen aortic root sections from the two groups (n = 5). The scale bars correspond to 500 μm. E Relative quantitative analysis of lipid deposition. F HE staining was performed on the two groups (n = 5). The scale bars correspond to 500 μm. G Relative quantitative analysis of aortic root lesion areas. H Masson staining was performed to assess the relative collagen content (n = 5). The scale bars correspond to 500 μm. I The relative collagen contents were quantified. J Representative sections double stained for CD68 (green) and Caspase-1/NLRP3/GSDMD (red). The scale bars correspond to 100 μm. K Western blot result of pyroptosis-related proteins with aortas of two groups (n = 3). The data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

Mice were randomly divided into four groups with different diets for 16 weeks: HFD group (fed with high fat western-type diet), HM groups (fed with a methionine-supplemented diet), Casp1^−/− group (fed with a high fat diet), Casp1^−/− group (fed with a methionine-supplemented diet). A HE staining was performed on the four groups (n = 5). The scale bars correspond to 500 μm. B Relative quantitative analysis of aortic root lesion areas. C Oil-red O staining was performed on frozen aortic root sections from the four groups (n = 5). The scale bars correspond to 500 μm. D Relative quantitative analysis of lipid deposition. E Representative images of Oil-red O staining of cardiac aorta in mice of four groups (n = 5). The scale bars correspond to 1 mm. F Representative images of Masson staining (n=5). The scale bars correspond to 500 μm. G The relative collagen contents were quantified. H Representative sections double stained for CD68 (green) and Caspase-1/GSDMD (red) (n = 5). The scale bars correspond to 100 μm. I Western blot result of pyroptosis-related proteins with aortas of four groups (n = 3). J–M Relative protein expressions quantification of pyroptosis-related proteins. N Plasma levels of IL-1β assessed by ELISA (n = 10). The data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

Hcy treatment triggers pyroptosis in THP-1. A Increases in the expression of pyroptosis-related genes (NLRP3 and IL-1β) at mRNA levels by different concentrations Hcy of 0, 1, 5, and 10 mM. B Western blot was used to evaluate the pyroptosis-related proteins level in different groups. C–G Relative protein expressions quantification of pyroptosis-related proteins. H Cell viability detected by CCK8. I The cell death was measured with Hoechst 33342 (blue)/PI (red) double-fluorescent staining. The scale bars correspond to 100 μm. J The levels of LDH were measured by commercial kits. K The morphology of cells was observed with scanning electron microscopy (SEM). The scale bars correspond to 10 μm. The data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

Caspase-1 inhibitor represses Hcy-induced macrophage pyroptosis. Macrophages were pretreated with caspase-1 inhibitor (VX-765, 10 μM) for 1 h, and then the cells were incubated with Hcy (10 mM) for 24 h. A Western blot was used to evaluate the pyroptosis-related proteins level in different groups. B–E Quantitative analysis of pyroptosis-associated protein expression. F Cell viability detected by CCK8. G The cell death was measured with Hoechst 33,342 (blue)/PI (red) double-fluorescent staining. The scale bars correspond to 100 μm. H LDH assay was used to evaluate the cell membrane integrity. I The morphology of cells was observed with scanning electron microscopy (SEM). The scale bars correspond to 10 μm. The data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

Pretreatment with ROS inhibitor suppresses Hcy-induced macrophage pyroptosis. Macrophages were pretreated with ROS inhibitor (NAC, 5 mM) for 1 h, and then the cells were incubated with Hcy (10 mM) for 24 h. A JC-1 assay was used to examine the mitochondrial membrane potential. The scale bars correspond to 100 μm. B Intracellular ROS level was detected using a DCFH-DA probe (green). Mitochondrial ROS level was detected by Mito-Sox Staining (red). The scale bars correspond to 100 μm. C Western blot was used to evaluate the pyroptosis-related proteins level in different groups. NAC pretreatment suppressed the upregulation of pyroptosis-associated protein levels in the presence of Hcy. D–G Quantitative analysis of pyroptosis-associated protein expression. H LDH assay was used to evaluate the cell membrane integrity. I The levels of ATP were measured by commercial kits after Hcy treated. The data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

Hcy induced ER stress response of macrophages. A Western blot was used to evaluate the ERS-related proteins (p-PERK, p-eif2α, ATF4, CHOP) level after Hcy treated. B–E Relative protein expressions quantification of ERS-related proteins. F Western blot was used to evaluate the pyroptosis-related proteins level after ERS inhibitor 4PBA pretreatment. G–M Relative protein expressions quantification of ERS-related and pyroptosis-associated proteins. The data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 7

Hcy increases ER-mitochondria colocalization. A Western blot was used to evaluate the MAM-related proteins (p-IP3R, VDAC, mfn-2) level after Hcy treated. B–D Relative protein expressions quantification of MAM-related proteins. E The ER was stained with ER-tracker (shown in green), and mitochondria were stained with mito-tracker (shown in red) and then imaged using confocal microscopy. Colocalization was shown in yellow in the merged images. The scale bars correspond to 30 μm. F Ca²⁺ change with the fluorescence. The levels of cellular and mitochondrial calcium were measured by Fluo-4, AM and Rhod-2, AM, respectively. The scale bars correspond to 100 μm. G Western blot was used to evaluate the pyroptosis-related protein level after calcium chelator BAPTA pretreatment. H–K Relative protein expressions quantification of pyroptosis-associated proteins. The data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 8

A model hypothesis diagram demonstrates the possible mechanism of Hcy promoting macrophage pyroptosis to accelerate atherosclerosis