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. 2024 Mar 8;9(5):e176065.
doi: 10.1172/jci.insight.176065.

CDK1 inhibition reduces osteogenesis in endothelial cells in vascular calcification

Yan Zhao  1 , Yang Yang  1 , Xiuju Wu  1 , Li Zhang  1 , Xinjiang Cai  1 , Jaden Ji  1 , Sydney Chen  1 , Abigail Vera  1 , Kristina I Boström  1   2 , Yucheng Yao  1
Affiliations

CDK1 inhibition reduces osteogenesis in endothelial cells in vascular calcification

Yan Zhao et al. JCI Insight. .

Abstract

Vascular calcification is a severe complication of cardiovascular diseases. Previous studies demonstrated that endothelial lineage cells transitioned into osteoblast-like cells and contributed to vascular calcification. Here, we found that inhibition of cyclin-dependent kinase (CDK) prevented endothelial lineage cells from transitioning to osteoblast-like cells and reduced vascular calcification. We identified a robust induction of CDK1 in endothelial cells (ECs) in calcified arteries and showed that EC-specific gene deletion of CDK1 decreased the calcification. We found that limiting CDK1 induced E-twenty-six specific sequence variant 2 (ETV2), which was responsible for blocking endothelial lineage cells from undergoing osteoblast differentiation. We also found that inhibition of CDK1 reduced vascular calcification in a diabetic mouse model. Together, the results highlight the importance of CDK1 suppression and suggest CDK1 inhibition as a potential option for treating vascular calcification.

Keywords: Cardiovascular disease; Cell biology; Vascular biology.

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Figures

Figure 1
Figure 1. CDK inhibitor AT7519 prevents endothelial lineage cells from osteogenic differentiation and reduces arterial calcification in Mgp–/– mice.
(A) Micro-CT imaging of VE-cadherincre/ERT2 RosatdTomato Mgp–/– mice and VE-cadherincre/ERT2 RosatdTomato mice after the administration of AT7519 (n = 6). Saline was used as control. Arrow and circles highlight the aortic calcification. Scale bars: 5 mm. (B) Alizarin red staining of descending aortas of VE-cadherincre/ERT2 RosatdTomato Mgp–/– mice after the administration of AT7519 (n = 6). Scale bars: 100 μm. (C) FACS analysis of aortic cells isolated from VE-cadherincre/ERT2 RosatdTomato Mgp–/– mice after the administration of AT7519 (n = 6). (D) Expression of the osteogenic markers osterix and osteopontin (OPN) and the endothelial markers eNOS and Flk1 in tdTomato+ aortic cells isolated from VE-cadherincre/ERT2 RosatdTomato Mgp–/– mice and VE-cadherincre/ERT2 RosatdTomato mice after the administration of AT7519, shown by real-time PCR (n = 6). Data in D were analyzed for statistical significance by 1-way ANOVA with Tukey’s multiple-comparison test. The bounds of the boxes are upper and lower quartiles with data points, the line in the box is the median, and whiskers are maximal and minimal values. ***P < 0.0001.
Figure 2
Figure 2. AT7519 prevents endothelial lineage cells from acquiring osteogenic capacity.
(A) Von Kossa staining and total calcium quantitation in osteogenesis assays using tdTomato+ aortic cells isolated from VE-cadherinCreERT2 RosatdTomato Mgp–/– mice treated with AT7519 (n = 9). VE-cadherinCreERT2 RosatdTomato mice were used as controls. Scale bars: 50 μm. (B) Tube formation assays using tdTomato+ aortic cells isolated from VE-cadherinCreERT2 RosatdTomato Mgp–/– mice treated with AT7519 (n = 9). VE-cadherinCreERT2 RosatdTomato mice were used as controls. Red, tdTomato. Scale bars: 50 μm. (C) Micro-CT images of ectopic bone formation with analysis of relative volume of bone formation and H&E staining after transplantation of tdTomato+ aortic cells isolated from VE-cadherinCreERT2 RosatdTomato Mgp–/– mice treated with AT7519 (n = 7). Saline was used as control. Scale bars: 5 mm (top) and 50 μm (bottom). (D) Laser Doppler perfusion images and immunostaining using anti-CD31 antibodies at ischemic sites after the transplantation of tdTomato+ aortic cells isolated from VE-cadherinCreERT2 RosatdTomato Mgp–/– mice treated with AT7519 (n = 8). Saline was used as control. Scale bars: 10 mm (top) and 50 μm (bottom). Data were analyzed for statistical significance by 1-way ANOVA with Tukey’s multiple-comparison test (A, B, and D) or unpaired, 2-tailed Student’s t test (C). In A–C, the bounds of the boxes are upper and lower quartiles with data points, the line in the box is the median, and whiskers are maximal and minimal values. Data in D are presented as mean ± SD. ***P < 0.0001.
Figure 3
Figure 3. Inhibition of CDK1 prevents osteogenic differentiation in MGP-depleted ECs.
(A) Expression of CDKs in tdTomato+ aortic cells isolated from VE-cadherincre/ERT2 RosatdTomato Mgp–/– mice and VE-cadherincre/ERT2 RosatdTomato mice, as determined by real-time PCR (n = 6). (B) Immunostaining of CDK1 in the aortic tissues of VE-cadherincre/ERT2 RosatdTomato Mgp–/– mice and VE-cadherincre/ERT2 RosatdTomato mice (n = 6). Scale bars: 50 μm. (C) Expression of MGP in HAECs after transfection of MGP siRNAs (n = 8). SCR, scramble siRNA. (D) Immunoblotting of CDK1, osteogenic, and endothelial markers in HAECs after transfection of MGP siRNAs and treatment with osteogenic induction media. Positive control (Pos Ctr) for CDK1 blotting: MCF-7 cells as verified by Thermo Fisher Scientific. Negative control (Neg Ctr) for CDK1 blotting: MCF-7 cells transfected with CDK1 siRNA. Positive control for osterix and osteopontin (OPN) blotting: MC3T3 osteoblast cells. Negative control for osterix and OPN blotting: HAECs. Positive control for eNOS and FLK1 blotting: HAECs. Negative control for eNOS and FLK1 blotting: MC3T3 osteoblast cells. Each lane represents an independent experimental group. (E) Immunoblotting of CDK1, OPN, and eNOS in HAECS after transfection of MGP siRNA in combination of with CDK1 siRNA and treatment with osteogenic induction media. Positive and negative controls are the same as in D. Each lane represents an independent experimental group. (F) Time-course expression of osteogenic and endothelial markers in HAECs after transfection of MGP siRNA and treatment with AT7519 or saline control (n = 3). Data were analyzed for statistical significance by unpaired, 2-tailed Student’s t test (A and C) or 1-way ANOVA with Tukey’s multiple-comparison test (F). In A and C, the bounds of the boxes are upper and lower quartiles with data points, the line in the box is the median, and whiskers are maximal and minimal values. Data in F are presented as mean ± SD. ***P < 0.0001.
Figure 4
Figure 4. Endothelial deletion of CDK1 prevents aortic endothelial lineage cells from undergoing osteogenic differentiation and reduces aortic calcification.
(A) Micro-CT imaging of VE-cadherincre/ERT2 Cdk1fl/fl Mgp–/– mice and VE-cadherincre/ERT2 Cdk1fl/fl mice after tamoxifen administration (n = 10). Saline was used as control. Arrow and circles highlight the calcification. Scale bars: 5 mm. (B) Alizarin red staining of descending aortas of VE-cadherincre/ERT2 Cdk1fl/fl Mgp–/– mice and VE-cadherincre/ERT2 Cdk1fl/fl mice after tamoxifen administration (n = 6). Scale bars: 100 μm. (C) Total aortic calcium and expression of Cdk1 and osteogenic and endothelial markers in CD31+CD45– aortic cells isolated from VE-cadherincre/ERT2 Cdk1fl/fl Mgp–/– mice and VE-cadherincre/ERT2 Cdk1fl/fl mice after tamoxifen administration (n = 6). Data in C were analyzed for statistical significance by 1-way ANOVA with Tukey’s multiple-comparison test. The bounds of the boxes are upper and lower quartiles with data points, the line in the box is the median, and whiskers are maximal and minimal values. ***P < 0.0001.
Figure 5
Figure 5. Limiting CDK1 upregulates ETV2 to prevent endothelial lineage cells from undergoing osteogenic differentiation.
(A and B) Expression of ETV2 in tdTomato+ aortic cells isolated from VE-cadherincre/ERT2 RosatdTomato Mgp–/– mice and VE-cadherincre/ERT2 RosatdTomato mice, as shown by real-time PCR (A) and immunoblotting (B) (n = 6). Positive control (Pos Ctr) for ETV2 blotting: Mouse ECs induced from embryonic stem cells (66). Negative control (Neg Ctr) for ETV2 blotting: HAECs transfected with ETV2 siRNA. Each lane represents an independent experimental group. (C) Expression of ETV2 in CD31+CD45– aortic cells isolated from VE-cadherincre/ERT2 Cdk1fl/fl Mgp–/– mice and VE-cadherincre/ERT2 Cdk1fl/fl mice after tamoxifen administration (n = 6). (D) ETV2 expression in HAECs after transfection of MGP siRNA in combination with AT5719 treatment (1–10 μM), as shown by real-time PCR (n = 6). (E) Immunoblotting of ETV2, osterix, osteopontin (OPN), eNOS, and FLK1 in HAECs after transfection of MGP siRNA in combination with infection of lentiviral vectors containing CMV-driven ETV2 cDNA and treatment with osteogenic induction media. Positive and negative controls are the same as described in B and Figure 3. Each lane represents an independent experimental group. (F and G) Micro-CT imaging and Alizarin red and von Kossa staining (F), and total aortic calcium with ETV2 expression (G) of descending aortas of Mgp–/– mice treated with adeno-associated viral (AAV) vectors containing CMV-promoter-driven ETV2 cDNA (n = 8). Empty vectors were used as controls. Scale bars: 5 mm (micro-CT) and 100 μm (staining). (H) Expression of osteogenic and endothelial markers in CD31+CD45– aortic cells from Mgp–/– mice treated with AAV vectors containing CMV-promoter-driven ETV2 cDNA (n = 8). Empty vectors were used as controls. Data were analyzed for statistical significance by 1-way ANOVA with Tukey’s multiple-comparison test (A, C, D, and H) or unpaired, 2-tailed Student’s t test (G). The bounds of the boxes are upper and lower quartiles with data points, the line in the box is the median, and whiskers are maximal and minimal values. ***P < 0.0001.
Figure 6
Figure 6. Limiting CDK1 reduces aortic calcification in diabetic Ins2Akita/+ mice.
(A and B) Alizarin red staining (A) and total aortic calcium (B) of descending aortas of Ins2Akita/+ mice after AT7519 administration (n = 10). Scale bars: 100 μm. (C) Immunoblotting of ETV2, osterix, and FLK1 in CD31+CD45– aortic cells isolated from Ins2Akita/+ mice after AT7519 administration. Positive and negative controls are the same as in Figures 3 and 5. Each lane represents an independent experimental group. (D) Micro-CT imaging of descending aortas of VE-cadherincre/ERT2 Cdk1fl/fl Ins2Akita/+ mice and VE-cadherincre/ERT2 Cdk1fl/fl mice after tamoxifen administration (n = 5). Scale bars: 50 μm. (E) Total aortic calcium and expression of CDK1 and ETV2 in CD31+CD45– aortic cells isolated from VE-cadherincre/ERT2 Cdk1fl/fl Ins2Akita/+ mice and VE-cadherincre/ERT2 Cdk1fl/fl mice after tamoxifen administration (n = 6). Data in B and E were analyzed for statistical significance by 1-way ANOVA with Tukey’s multiple-comparison test. The bounds of the boxes are upper and lower quartiles with data points, the line in the box is median, and whiskers are maximal and minimal values. **P < 0.001; ***P < 0.0001.
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