Cigarette Tar Enhanced ECs Pyroptosis via CAMKII/Drp1/mtDNA: Novel Insight Into the Mechanism of Plaque Erosion

Abstract

Smoking is the only cardiovascular risk factor for plaque erosion. We found cigarette tar resulted in erosion-like lesion development in apolipoprotein E^-/- mice, with mural thrombosis, discontinuous endothelium, platelet activation, smooth muscle cell proliferation, and hyaluronic acid accumulation in the aorta. Single-cell transcriptomics revealed that genes relating to pyroptosis, platelet activation, and leukocytes adhesion were significantly increased in an endothelial cell subset. Rescue assays indicated cigarette tar caused human coronary artery endothelial cell pyroptosis by enhanced calcium-calmodulin-dependent protein kinase II / dynamin-related protein 1-mediated mitochondrial fission and mitochondrial DNA release via activating Ca²⁺ signaling. Inhibition of endothelial cell pyroptosis may be a novel therapeutic strategy to reduce plaque erosion.

Keywords: cigarette tar; endothelial cells; plaque erosion; pyroptosis.

Graphical abstract

Figure 1

Cigarette Tar Caused an Eroded-like Plaque Animal Model in ApoE^−/− Mice Apolipoprotein E (ApoE)^−/− mice were intraperitoneally injected with 150 mg/kg/d cigarette tar for 12 weeks. (A) The survival of ApoE^−/− mice at 12 weeks; n = 30. (B) Evans blue staining; n = 11. (C) Immunofluorescence staining of mouse aorta; n = 10 per group. (D) Typical picture of mural thrombus in mice. The red arrows represent mural thrombus. (E,F) Hematoxylin and eosin staining for mouse carotid artery, n = 10 per group, and thrombus sample, n = 5. (G) The intimal detachment of mouse aorta and platelet activation were observed by scanning electron microscopy. The red arrows represent endothelial detachment and activated platelets; n = 10. (H,I) The messenger RNA (mRNA) level of von Willebrand factor (vWF) and tissue factor (TF) in the mouse aorta; n = 10. (J,K) Immunofluorescence staining of mouse arteries with TF and vWF; n = 10. Results are expressed as mean ± SD. Comparisons of parameters were performed with Student’s t-test (B,C,H,I,K), log-rank test (A), or chi-square test (D). ∗∗∗P < 0.001. CTRL = control; DAPI = 4ʹ,6-diamidino-2-phenylindole; Tie2 = tyrosine-protein kinase receptor TEK; α-SMA = α- smooth muscle actin.

Figure 2

Cigarette Tar Caused HCAEC Pyroptosis In Vivo and In Vitro Human coronary artery endothelial cells (HCAECs) were treated with 0-100 μg/mL tar for 12, 24, or 48 hours. (A) Cell counting kit-8 assay was used to analyze cell viability; n = 5. ∗∗P < 0.01 vs 0 μg/mL. (B) Western blot was used to evaluated the protein level of NLRP3, caspase 1, and GSDMD; n = 4. (C) Scanning electron microscopy was used to observe cell swelling; n = 5. (D) Flow cytometry was used to evaluate cell viability; n = 5. (E) Tie2/GSDMD immunofluorescence staining for mouse aorta; n = 10. (F,G) The level of interleukin-1β and interleukin-18 in mouse serum; n = 10. (H,I) The level of cell-free DNA and myeloperoxidase (MPO) in mouse serum; n = 10 per group. Results are expressed as mean ± SD. Comparisons of parameters were performed with Student’s t-test (E-I) or 1-way analysis of variance with Bonferroni post hoc test (A,B,C,D). ∗∗P < 0.01, ∗∗∗P < 0.001. Comp-FITC-A = annexin V; Comp-PE-A = PI ; FL = full-length; other abbreviations as in Figure 1.

Figure 3

Caspase1 Deletion Diminished Cigarette Tar–Induced Eroded Plaque (A) The survival of the mice; n = 30. (B) Typical thrombus of a mouse artery. (C) The intimal detachment of mouse artery was observed by electron microscope; n = 5. (D) Evans blue staining of mouse arteries; n = 10. (E,F) Tie2 staining; n = 10. (G) Costaining of Tie2 and GSDMD in mouse artery; n = 10. (H,I) The mRNA levels of VCAM1 and E-selectin in mouse artery; n = 10. (J-L) Serum MPO, interleukin-1β, and interleukin-18 levels in ApoE^−/−Caspase 1^−/− and ApoE^−/− mice; n = 10. (M,N) The mRNA levels of TF and vWF in mouse artery; n = 10. (O) Immunofluorescence staining for TF and vWF; n = 10. Results are expressed as mean ± SD. Comparisons of parameters were performed with Student’s t-test (D), log-rank (A) 1-way analysis of variance with Bonferroni post hoc test (F,G,J-N), or chi-square test (B). ∗P < 0.05, ∗∗∗P < 0.001. Abbreviations as in Figures 1 and 2.

Figure 4

Cigarette Tar Enhanced Mitochondrial Fission via Drp1 Activation (A) HCAECs were treated with cigarette tar with 100 μg/mL for 6 hours. The cell lysates were analyzed by tandem mass tagged proteomics and the differential proteins were indicated by heat maps; n = 3. (B) The level of dynamin-related protein 1 (Drp1) and p-Drp1^ser616; n = 3. (C,D) Mito-tracker staining for mitochondrial fraction and scanning electron microscopy; n = 5. (E) The level of Drp1 in mitochondrial and cytoplasm lysates; n = 3. (F) Costaining of Tomm20 and Drp1 in HCAECs; n = 5. (G) The HCAECs were treated with 5 μmol/L Mdivi-1 for 1 hours and cigarette tar with 100 μg/mL for 24 hours, the protein levels of NLRP3, caspase 1 and GSDMD were detected; n = 3. Results are expressed as mean ± SD. Comparisons of parameters were performed with Student’s t-test (A,B,E,F) or 1-way analysis of variance with Bonferroni post hoc test (G). ∗p<0.05 and ∗∗p<0.01. COX IV = cytochrome c oxidase subunit IV; TEM = transmission electron microscope; other abbreviations as in Figures 1 and 2.

Figure 5

Cigarette Tar Promoted Drp1-Mediated Mitochondrial Fission via Activating Ca²⁺/CAMKII Pathway (A,B) Flow cytometry and immunofluorescence were used to evaluate the fluorescence intensity of intracellular calcium ions; n = 5. (C) The endothelial cells were pretreated with 5 μmol/L of KN93 and 5 μmol/L of 1,2-bis(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA) before receiving 100 μg/mL tar. The calcium–calmodulin dependent protein kinase II (CAMKII) and Drp1 level in mitochondrial were detected; n = 4. (D) The endothelial cells were pretreated with 5 μmol/L of KN93 and 5 μmol/L of BAPTA before receiving 100 μg/mL tar. The level of p-CAMKII and p-Drp1^ser616 were detected; n = 4. (E) Mito-tracker staining; n = 5. (F,G) Costaining of mito-tracker/Drp1 and Drp1/CAMKII in endothelial cells; n = 5. (H) The level of NLRP3, caspase 1, and GSDMD were detected; n = 3. (I) Calcium-free medium: The cells were cultured in a medium free of calcium ions. 2-Aminoethyl diphenylborinate (2-APB): pretreated with 5 μmol/L of 2-APB for 30 minutes. Flow cytometry was used to assess intracellular calcium ion levels; n=5. (J) The level of NLRP3, caspase 1, and GSDMD were detected; n = 4. Results are expressed as mean ± SD. Comparisons of parameters were performed with 1-way analysis of variance with Bonferroni post hoc test. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. MFI = mean fluorescent intensity; mtDNA = mitochondrial DNA; P2 = the proportion of calcium ion fluorescence-positive cells; other abbreviations as in Figures 1, 2, and 4.

Figure 6

Cigarette Tar Promoted NLRP3 Inflammasome Activation and Pyroptosis via Enhancing mtDNA Release (A) HCAECs were treated with 100 μg/mL cigarette tar for 24 hours, with or without 5 μmol/L Mdivi-1 for 1 hour pretreatment. The short regions of the D-Loop genes were used to quantify mtDNA and nuclear DNA, respectively, from which the mtDNA/nuclear DNA ratio was calculated to determine the mtDNA copy number. The cytosol mtDNA copy number; n = 5. (B) The mtDNA copy number; n = 6. (C) Costaining of double-stranded DNA (dsDNA) and mito-tracker in HCAECs; n = 5. (D) Costaining of dsDNA and mito-tracker in artery of ApoE^−/− mice; n = 10. (E) Cell-free mtDNA level in mice serum; n = 6. (F) Ethidium bromide (Et-Br) was used to establish mtDNA-deletion cell line. Cell counting kit-8 (CCK-8) assay was used to determine the cell viability; n = 6. (G) Annexin V–propidium iodide (PI) was used to analyze cell death; n = 5. (H) Expression of NLRP3, GSDMD, and caspase 1; n = 3. (I,J) Costaining of dsDNA and NLRP3; n = 5. (K) Antibodies to NLRP3 were used to purify the protein. After the lysate was separated, mtDNA (D-Loop) levels in the lysate were detected by polymerase chain reaction; n = 6. (L) Short hairpin negative control (sh-NC) and sh-NLRP3 were transfected into cells. The mtDNA (being pretreated with or without deoxyribonuclease [DNase]) was extracted and transfected into cells (2 μg/mL). The expression of NLRP3, caspase 1, and GSDMD were evaluated; n = 3. (M) CCK-8 assay was used to determine the cell viability in HCAECs with sh-NC; n = 6. (N) Annexin V-PI was used to analyze cell death; n = 5. (O) CCK-8 assay was used to determine the cell viability in HCAECs with sh-NLRP3; n = 5. Results are expressed as mean ± SD. Comparisons of parameters were performed with 1-way analysis of variance with Bonferroni post hoc test. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. NS = not significant; other abbreviations as in Figures 1, 2, and 5.

Figure 7

Drp1 Knockdown–Attenuated Cigarette Tar Induced HCAEC Pyroptosis and mtDNA Release Adeno-associated virus (AAV)-Sh-NC group: mice were injected with AAV-NC virus. AAV-Sh-Drp1 group: mice were injected with AAV-Drp1 virus through a tail vein. (A) The Drp1 level in the mouse artery; n = 12. (B) Evans blue staining; n = 10. (C) Costaining of dsDNA and Tomm20 in artery of ApoE^−/− mice; n = 10. (D) Costaining of Tie2, GSDMD, and caspase 1 in artery of ApoE^−/− mice; n = 10. (E-H) Serum level of cell-free DNA, MPO, interleukin-18, and interleukin-1β; n = 10. Results are expressed as mean ± SD. Comparisons of parameters were performed with Student’s t-test. ∗∗P < 0.01 and ∗∗∗P < 0.001. Abbreviations as in Figures 1, 2, 4, and 6.