CypD induced ROS output promotes intracranial aneurysm formation and rupture by 8-OHdG/NLRP3/MMP9 pathway

Abstract

Reactive Oxygen Species (ROS) are widely accepted as a pernicious factor in the progression of intracranial aneurysm (IA), which is eminently related to cell apoptosis and extracellular matrix degradation, but the mechanism remains to be elucidated. Recent evidence has identified that enhancement of Cyclophilin D (CypD) under stress conditions plays a critical role in ROS output, thus accelerating vascular destruction. However, no study has confirmed whether cypD is a detrimental mediator of cell apoptosis and extracellular matrix degradation in the setting of IA development. Our data indicated that endogenous cypD mRNA was significantly upregulated in human IA lesions and mouse IA wall, accompanied by higher level of ROS, MMPs and cell apoptosis. CypD^-/- remarkably reversed vascular smooth muscle cells (VSMCs) apoptosis and elastic fiber degradation, and significantly decreased the incidence of aneurysm and ruptured aneurysm, together with the downregulation of ROS, 8-OHdG, NLRP3 and MMP9 in vivo and vitro. Furthermore, we demonstrated that blockade of cypD with CsA inhibited the above processes, thus preventing IA formation and rupture, these effects were highly dependent on ROS output. Mechanistically, we found that cypD directly interacts with ATP5B to promote ROS release in VSMCs, and 8-OHdG directly bind to NLRP3, which interacted with MMP9 to increased MMP9 level and activity in vivo and vitro. Our data expound an unexpected role of cypD in IA pathogenesis and an undescribed 8-OHdG/NLRP3/MMP9 pathway involved in accelerating VSMCs apoptosis and elastic fiber degradation. Repressing ROS output by CypD inhibition may be a promising therapeutic strategy for prevention IA development.

Keywords: Cyclophilin D; Intracranial aneurysm; Reactive oxygen species; Vascular smooth muscle cell.

Graphical abstract

Fig. 1

Presence of cypD mRNA in human intracranial aneurysm tissues A, Heatmap showing the transcript expression levels of 2337 differentially expressed genes (DEGs) in GSE75436 of 15 intracranial aneurysm tissues of human and their matched normal superficial temporal arteries. B, Volcano plot simultaneously demonstrating the fold changes (UIA/control) and Wilcoxon test results of 2337 DEGs. Red dots show the 1254 genes that were signifcantly upregulated. Blue dots show the 1083 genes that signifcantly downregulated. Specifc genes of interest are labeled with green dots. Abbreviations: ppif stands for cypD. C, Gene ontology (GO) analysis of upregulated DEGs in human IA tissues showing the diversified highly expressed biologic processes involved in oxidative stress, inflammation, apoptosis and ECM organization. D, Heat map showing that the differential expressions of specific upregulated DEGs related to cypD, ROS, inflammation, apoptosis and ECM degradation between IA tissues and their matched normal superficial temporal arteries. n = 15 vs n = 15.

Fig. 2

Typical images of intracranial aneurysm (IA) model A, Schematic diagram of intracranial aneurysm mouse molding method. B, Representative photographs of cerebral vascular in the bottom of mice brains. Left panel showing the normal cerebral vessels; middle panel shows the intracranial aneurysm (IA) of mice, the black arrow indicates unruptured aneurysm; right panel shows the ruptured IA of mice, the white arrow indicates ruptured aneurysm. C, Typical images of bleeding grades in ruptured aneurysms.

Fig. 3

Presence of cypD in intracranial aneurysm tissues from mouse A, Representative microphotographs of FISH staining for cypD mRNA (red) co-localization on VSMCs (SM22-α, green) in normal cerebral vessels and IA lesion. B, Representative images of immunohistochemical staining for cypD in normal cerebral vessels and IA lesion. C-D, Representative images of DHE staining (red) and in situ zymography staining (green) in normal cerebral vessels and IA lesion. E, Representative pictures of scanning electron microscope (SEM) and transmission electron microscope (TEM) in normal cerebral vessels and IA lesion. Upper panel shows the morphology of normal vessel and IA; lower panel shows the vascular wall structure of normal cerebral vessels and IA. SMC = smooth muscle cell, AS = apoptosis smooth muscle cell, EL = elastic fibre, EC = endothelial cell.

Fig. 4

Reduced the development of intracranial aneurysm (IA) in cypD KO mice A, The incidence of IA and ruptured IA in WT mice and cypD KO mice; B, The cumulative symptom-free survival in two groups. *P < 0.05. C, Blood pressure results in both groups. D, Representative images of hematoxylin-eosin (HE) staining and elastic fiber satining in different groups. n = 4 per group. E, Representative microphotographs and quantitative analyses of TUNEL staining (red) co-localization on VSMCs (SM22-α, green) in different groups. n = 4 per group. Bars represent mean ± SD. ns stands for no significance; *P < 0.05 vs. WT group; #P < 0.05 vs. WT + IA group.

Fig. 5

Downregulation of 8-OHdG/NLRP3/MMP9 pathway in cypD KO mice with IA A-F, Representative microphotographs of double immunofluorescence staining for 8-OHdG (red), NLRP3 (green) and MMP9 (red) in the VSMCs in diverse groups. n = 4 per group. Bars represent mean ± SD. ns stands for no significance; *P < 0.05 vs. WT group; #P < 0.05 vs. WT + IA group.

Fig. 6

CypD bonds together with ATP5B to release ROS in cerebral artery VSMCs A, Representative picture of immunofluorescence staining for primary VSMCs (SM22-α, green). B, Cell lysates were collected and immunoprecipitated with anti-ATP5B Ab and anti-IgG Ab, then western blotted with anti-ATP5B Ab and anti-cypD Ab. C-D, Representative picture and quantitative analyses of ROS staining (green) in diverse groups. n = 4 per group. Bars represent mean ± SD. *P < 0.05 vs. PBS + WT group; #P < 0.05 vs. H₂O₂+WT group. E,I, Representative dot blot images and quantitative analyses of 8-OHdG in three groups. n = 4 per group. Bars represent mean ± SD. *P < 0.05 vs. PBS group; #P < 0.05 vs. H₂O₂+WT group. E-H, Representative Western blot images and quantitative analyses of NLRP3 and MMP9 in three groups. n = 4 per group. Bars represent mean ± SD. *P < 0.05 vs. PBS group; #P < 0.05 vs. H₂O₂+WT group.

Fig. 7

NLRP3 binds to 8-OHdG and MMP9 in vascular smooth muscle cell of IA A, Representative picture of multiple immunofluorescence staining shows that 8-OHdG (red), NLRP3 (green), and MMP9 (red) were co-localized in smooth muscle cells (purple) of IA wall at mice. B, Cell lysates were collected and immunoprecipitated with anti-NLRP3 Ab and anti-IgG Ab, then western blotted with anti-NLRP3 Ab and anti-MMP9 Ab. C, Cell lysates were collected and immunoprecipitated with anti-NLRP3 Ab and anti-IgG Ab, then dot blotted with anti-8-OHdG Ab.

Fig. 8

CsA treatment reduced the development of intracranial aneurysm in wild-type (WT) mice A, The incidence of IA and ruptured IA in IA group and IA mice treatment with CsA; B, The cumulative symptom-free survival in both groups. *P < 0.05. C, Blood pressure results in tow groups. D-E, Representative images of hematoxylin-eosin (HE) staining and elastic fiber satining in different groups. n = 4 per group. F-G, Representative microphotographs and quantitative analyses of TUNEL staining (red) co-localization on VSMCs (SM22-α, green) in different groups. n = 4 per group. Bars represent mean ± SD. ns stands for no significance; *P < 0.05 vs. Normal group; #P < 0.05 vs. IA group.

Fig. 9

CsA treatment down-regulates 8-OHdG/NLRP3/MMP9 pathway in wild-type (WT) mice with IA A-F, Representative microphotographs of double immunofluorescence staining for 8-OHdG (red), NLRP3 (green) and MMP9 (red) in the VSMCs in diverse groups. n = 4 per group. Bars represent mean ± SD. ns stands for no significance; *P < 0.05 vs. Normal group; #P < 0.05 vs. IA group.

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