Silicate ions as soluble form of bioactive ceramics alleviate aortic aneurysm and dissection

Abstract

Aortic aneurysm and dissection (AAD) are leading causes of death in the elderly. Recent studies have demonstrated that silicate ions can manipulate multiple cells, especially vascular-related cells. We demonstrated in this study that silicate ions as soluble form of bioactive ceramics effectively alleviated aortic aneurysm and dissection in both Ang II and β-BAPN induced AAD models. Different from the single targeting therapeutic drug approaches, the bioactive ceramic derived approach attributes to the effect of bioactive silicate ions on the inhibition of the AAD progression through regulating the local vascular microenvironment of aorta systematically in a multi-functional way. The in vitro experiments revealed that silicate ions did not only alleviate senescence and inflammation of the mouse aortic endothelial cells, enhance M2 polarization of mouse bone marrow-derived macrophages, and reduce apoptosis of mouse aortic smooth muscle cells, but also regulate their interactions. The in vivo studies further confirm that silicate ions could effectively alleviate senescence, inflammation, and cell apoptosis of aortas, accomplished with reduced aortic dilation, collagen deposition, and elastin laminae degradation. This bioactive ceramic derived therapy provides a potential new treatment strategy in attenuating AAD progression.

Keywords: Aortic aneurysm and dissection; Cell apoptosis; Inflammation; Senescence; Silicate ions.

Graphical abstract

Fig. 1

Silicate ions as soluble form of bioactive ceramics alleviate senescence of MAECs. (A) SA-β-Gal (blue, senescence marker) staining and quantitative analysis of SA-β-Gal staining (presented as the percentage of SA-β-Gal positive cells) in cultured MAECs treated with Ctrl (n = 6) or CS (n = 6) for 72 h. (B) Representative Western blots of SIRT1 (senescence-associated protein) and quantitative analysis of protein levels presented as mean ratio values quantified from protein bands of SIRT1 versus GAPDH (an endogenous control for protein expression) in cultured MAECs treated with Ctrl (n = 5) or CS (n = 5) for 72 h. (C) Representative Western blots of γH2A.X (DNA damage-associated protein) and quantitative analysis of protein levels presented as mean ratio values quantified from protein bands of γH2A.X versus GAPDH in cultured MAECs treated with Ctrl (n = 6) or CS (n = 7) for 72 h. (D) Representative immunofluorescence images of γH2A.X (red)/DAPI (blue) and quantitative analysis of γH2A.X nuclear expression presented as the percentage of γH2A.X positive cells in cultured MAECs treated with Ctrl (n = 5) or CS (n = 5) for 72 h. The top left corner images show the γH2A.X staining of MAECs. Scale bars, 100 μm (A) and 50 μm (D). MAECs, mouse aorta endothelial cells. Ctrl, control medium. CS, silicate ions containing medium. All data are presented as mean ± SEM. Statistical analysis was performed using unpaired Student's t-test, **P < 0.01.

Fig. 2

Silicate ions as soluble form of bioactive ceramics alleviate inflammation of MAECs and MBMMs. (A) Representative Western blot of acetylated p65 (inflammation-associated protein, Ac-p65) and quantitative analysis of protein levels presented as mean ratio values quantified from protein bands of Ac-p65 versus total p65 (Total-p65) in cultured MAECs treated with Ctrl (n = 3) or CS (n = 3) for 72 h. (B) Representative Western blots of ICAM1 and VCAM1 (inflammation-associated proteins) and their quantitative analysis of protein levels represented as mean ratio values quantified from protein bands of ICAM1 and VCAM1 versus GAPDH in cultured MAECs treated with Ctrl, LPS or LPS + CS (n = 5). (C) Representative flow cytometric analysis of MBMM polarization and quantitative analysis of MBMM polarization (presented as the percentage of CD206⁺ F4/80⁺ cells) treated with Ctrl (n = 4) or CS (n = 4) for 72 h using phenotypic markers F4/80 (M0) and CD206 (M2). (D) The secreted TGF-β levels detected by Elisa assay in cultured MBMM conditioned medium treated with Ctrl (n = 4) or CS (n = 4) for 72 h. (E) Schematic diagram of direct contact coculture model: MAECs were pretreated with Ctrl or CS for 72 h, then MBMMs were added and co-cultured with MAECs for 1 h. (F) Representative images and quantitative cell count of THP-1 adhered to MAECs pretreated with Ctrl, LPS or LPS + CS in direct contact co-culture model. (n = 3). Scale bar, 100 μm. (G) Schematic diagram of indirect contact co-culture model: MAECs were pretreated with Ctrl or CS for 72 h, then the conditioned medium was used to culture MBMMs for 48 h. (H) Representative flow cytometric images and quantitative analysis of MBMM polarization (presented as the percentage of CD206⁺ F4/80⁺ cells) treated with conditioned medium from Ctrl pretreated MAECs or CS pretreated MAECs (n = 4) using phenotypic markers F4/80 (M0) and CD206 (M2) in indirect contact co-culture model. MBMMs, mouse bone marrow-derived macrophages. All data are presented as mean ± SEM. Statistical analysis was performed using (A, C, E, G and H) unpaired Student's t-test, (B and F) one-way ANOVA *P < 0.05 or **P < 0.01 vs. Ctrl, ^#P < 0.01 vs. LPS. ns: no significant difference.

Fig. 3

Silicate ions as soluble form of bioactive ceramics reduce MASMC apoptosis. (A) Flow cytometric quantification analysis of apoptosis in MASMCs after treatment with Ctrl, Ang II or Ang II + CS using Annexin V-FITC and PI. (n = 7) (B) Representative Western blots of Cleaved-Cas3 and Cleaved-Cas9 (apoptotic-associated proteins) and their quantitative analysis of protein levels presented as mean ratio values quantified from protein bands of Cleaved-Cas3 and Cleaved-Cas9 versus GAPDH in cultured MASMCs treated with Ctrl (n = 5) or CS (n = 5) for 72 h. (C) Schematic diagram of direct contact co-culture model: MASMCs were pretreated with Ctrl or CS for 72 h, then MBMMs were added and cocultured with MASMCs for 1 h. (D) Representative images and quantitative cell counts of MBMMs adhered to MASMCs pretreated with Ctrl (n = 6) or CS (n = 6) in direct contact co-culture model. Scale bar, 100 μm. (E) Schematic diagram of indirect contact co-culture model: MBMMs were pretreated with Ctrl or CS for 72 h, then the conditioned medium was used to culture MASMCs for 48 h. (F) Representative flow cytometric images and quantitative analysis of MASMC apoptosis (presented as the percentage of Annexin V⁺ cells) treated with conditioned medium from Ctrl pretreated MBMMs (Ctrl-MBMMs, n = 5) or CS pretreated MBMMs (CS-MBMMs, n = 5) using apoptotic markers Annexin V-FITC and PI in indirect contact co-culture model. MASMCs, mouse aorta smooth muscle cells. All data are presented as mean ± SEM. Statistical analysis was performed using (A) one-way ANOVA, (B–F) unpaired Student's t-test, **P < 0.01, ***P < 0.001 vs.Ctrl, ^#P < 0.01 vs. Ang II.

Fig. 4

Silicate ions as soluble form of bioactive ceramics reduce the aortic dilation and collagen deposition of abdominal aorta in the Ang II-induced AAD model. (A) Schematic diagram of experimental design: 49-day-old mice were fed a high-fat diet and then implanted with osmotic pumps containing Ang II (1.44 mg/kg/day) or PBS at day 56 for 28 days. Silicate ions-containing saline or saline control were intravenously injected into mice 7 times (every other day) after 7 days' Ang II or PBS infusion. Experimental groups were designated as Ang II + Saline, and Ang II + CS, respectively, and PBS pumping plus saline injection were used as control (PBS + Saline). (B) The mortality rate and AAD incidence rate after different treatments (n = 5 for PBS + Saline, n = 11 for Ang II + Saline and Ang II + CS). (C) Representative photographs of the whole aortas after different treatments. Red arrows show AADs. (D) Representative ultrasound (US) images and quantified maximal diameters of abdominal aortas. White lines and values represent the maximal diameters of abdominal aortas (n = 4 for PBS + Saline, n = 5 for Ang II + Saline, and n = 6 for Ang II + CS). (E) Representative HE and Masson's trichrome staining images and summary collagen deposition score calculated from the Masson's trichrome staining (n = 4 for PBS + Saline, n = 5 for Ang II + Saline, and n = 6 for Ang II + CS). Scale bars, 5 mm (C), 1 mm (D) and 200 μm (E). All data are presented as mean ± SEM, one-way ANOVA, ***P < 0.001 vs. PBS + Saline, ^##P < 0.01 vs. Ang II + Saline.

Fig. 5

Silicate ions as soluble form of bioactive ceramics reduce senescence, inflammation, and cell apoptosis of the aortas in the Ang II-induced AAD model. (A) Representative images of SA-β-gal positive staining (blue) and quantitative results (presented as percentage SA-β-gal positive area versus total area) in abdominal aortas. Red boxed areas are expanded to show representative high-power fields (n = 4 for PBS + Saline, n = 5 for Ang II + Saline and Ang II + CS). (B) Representative immunohistochemistry images and summary scores of inflammation-associated proteins ICAM1 and VCAM1 in abdominal aortas after different treatments (n = 4 for PBS + Saline, n = 3 for Ang II + Saline, and n = 5 for Ang II + CS). (C) Representative immunohistochemistry images and summary scores of caspase 3 and caspase 9 (apoptosis-associated proteins) in abdominal aortas (n = 3 for PBS + Saline and Ang II + Saline, and n = 4 for Ang II + CS). Scale bars, 200 μm (A), and 100 μm (B and C). All data are presented as mean ± SEM, one-way ANOVA. *P < 0.05, **P < 0.01 or ***P < 0.001 vs. PBS + Saline, ^#P < 0.05 or ^##P < 0.01 vs. Ang II + Saline.

Fig. 6

Silicate ions as soluble form of bioactive ceramics administrated in the mode of Design I suppress β-BAPN induced AAD formation: reducing mortality rate and AAD incidence rate as well as inhibiting the aortic dilation and elastin laminae degradation of the aortic arches. (A) Schematic diagram of treatments in Design I: 21-day-old mice were administrated with β-BAPN (0.8 g/kg/day) containing water or free water for 28 days. Silicate ions-containing saline or saline control were intravenously injected in mice 7 times (every other day) after 7 days' β-BAPN administration. Experimental groups were designated as β-BAPN + Saline and β-BAPN + CS, respectively, and pure water feeding plus saline injection were used as control (H₂O + Saline). (B) The mortality rate and AAD incidence rate after different treatments (n = 5 for H₂O + Saline, n = 13 for β-BAPN + Saline and β-BAPN + CS). (C) Representative photographs of whole aortas in Design I. Red arrows show AADs. (D) Representative US images and quantitative maximal diameters of aortic arches in Design I. White lines and values represent the maximal diameters of aortic arches (n = 5 for H₂O + Saline, β-BAPN + Saline, and β-BAPN + CS). (E) Representative photographs and quantification of the percentage of different degrees of the elastin laminae degradation in aortic aortas using modified Gomori staining in Design I. Red boxed areas are expanded to show representative high-power fields. Elastin laminae gradation grades: intact internal elastic lamina (I), mild elastin fragmentation (II), severe elastin digestion (III), and severe elastin digestion with a visible ruptured site (IV). (n = 3 for H₂O + Saline, n = 6 for β-BAPN + Saline and β-BAPN + CS). Scale bars, 5 mm (C), 2 mm (D) and 500 μm (E). Data are presented as mean ± SEM, one-way ANOVA, ***P < 0.001 vs. H₂O + Saline, ^##P < 0.01 vs. β-BAPN + Saline.

Fig. 7

Silicate ions as soluble form of bioactive ceramics administrated in the mode of Design II suppress β-BAPN induced AAD formation: reducing mortality rate and AAD incidence rate as well as inhibiting the aortic dilation and collagen deposition of the aortic arches. (A) Schematic diagram of treatments in Design II: 21-day-old mice were administrated with β-BAPN (0.8 g/kg/day) containing water or free water for 28 days. Silicate ions-containing saline or saline control were intravenously injected in mice 14 times every day after 7 days' β-BAPN administration. Experimental groups were designated as β-BAPN + Saline and β-BAPN + H-CS, respectively, and pure water feeding plus saline injection were used as control (H₂O + Saline). (B) The mortality rate and AAD incidence rate after different treatment (n = 6 for H₂O + Saline, n = 18 for β-BAPN + Saline, and n = 13 for β-BAPN + CS). (C) Representative photographs of whole aortas in Design II. Red arrows show AADs. (D) Representative US images and quantitative maximal diameters of aortic arches in Design II. White lines and values represent the maximal diameters of aortic arches (n = 5 for H₂O + Saline, β-BAPN + Saline, and β-BAPN + CS). (E) Representative HE and Masson's trichrome staining images and summary collagen deposition score of aortic arches (n = 4 for H₂O + Saline, n = 4 for β-BAPN + Saline, and n = 6 for β-BAPN + CS). Scale bars, 2 mm (C), 1 mm (D) and 200 μm (E). All data are presented as mean ± SEM, one-way ANOVA. ***P < 0.001 vs. PBS + Saline, ^#P < 0.05 or ^##P < 0.01 vs. β-BAPN + Saline.

Fig. 8

Scheme describing the “silicate ion therapy” on AAD. (A) Intravenous injection of silicate ions as soluble form of bioactive ceramics attenuates both Ang II and β-BAPN induced AAD progression, including reduced mortality rate, AAD incidence rate, aortic dilation, collagen deposition and elastin degradation. (B) Silicate ions as soluble form of bioactive ceramics modulate the vascular microenvironment by inhibiting senescence, inflammation, and apoptosis of aorta. (C) Silicate ions as soluble form of bioactive ceramics alleviate MAEC senescence and inflammation, promote polarization of MBMMs towards M2 phenotype, inhibit the adhesion of MBMMs to MAECs (or MASMCs) and the apoptosis of MASMCs directly or by mediating cell-cell crosstalk.