J Korean Neurosurg Soc.  2023 Nov;66(6):642-651. 10.3340/jkns.2023.0005.

Cyclin-Dependent Kinase Inhibitor 2A is a Key Regulator of Cell Cycle Arrest and Senescence in Endothelial Colony-Forming Cells in Moyamoya Disease

• Choi SA
• Moon YJ
• Koh EJ
• Phi JH
• Lee JY
• Kim KH
• Kim SK

• KMID: 2547458
• DOI: http://doi.org/10.3340/jkns.2023.0005

Abstract

Objective
: Endothelial colony-forming cells (ECFCs) have been reported to play an important role in the pathogenesis of moyamoya disease (MMD). We have previously observed stagnant growth in MMD ECFCs with functional impairment of tubule formation. We aimed to verify the key regulators and related signaling pathways involved in the functional defects of MMD ECFCs.
Methods
: ECFCs were cultured from peripheral blood mononuclear cells of healthy volunteers (normal) and MMD patients. Low-density lipoproteins uptake, flow cytometry, high content screening, senescence-associated β-galactosidase, immunofluorescence, cell cycle, tubule formation, microarray, real-time quantitative polymerase chain reaction, small interfering RNA transfection, and western blot analyses were performed.
Results
: The acquisition of cells that can be cultured for a long time with the characteristics of late ECFCs was significantly lower in the MMD patients than the normal. Importantly, the MMD ECFCs showed decreased cellular proliferation with G1 cell cycle arrest and cellular senescence compared to the normal ECFCs. A pathway enrichment analysis demonstrated that the cell cycle pathway was the major enriched pathway, which is consistent with the results of the functional analysis of ECFCs. Among the genes associated with the cell cycle, cyclin-dependent kinase inhibitor 2A (CDKN2A) showed the highest expression in MMD ECFCs. Knockdown of CDKN2A in MMD ECFCs enhanced proliferation by reducing G1 cell cycle arrest and inhibiting senescence through the regulation of CDK4 and phospho retinoblastoma protein.
Conclusion
: Our study suggests that CDKN2A plays an important role in the growth retardation of MMD ECFCs by inducing cell cycle arrest and senescence.

Keyword

Moyamoya disease; Endothelial colony-forming cells; Cyclin dependent kinase inhibitor 2A; Cell cycle; Cellular senescence

Figure

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  Fig. 1. Cell growth arrest and senescence of MMD ECFCs. A : Cell proliferation is assessed via a high-throughput screening (HCS) assay. Representative growth curves (nonexpanded cells) of ECFCs are generated from cell images taken every 4 hours using the imaging system and its cell density algorithm. B : Doubling time results suggest that MMD ECFCs are significantly slower than normal ECFCs. C : Cell cycle analysis by flow cytometry shows that G1 arrest is higher in MMD ECFCs than in normal ECFCs. D and E : Senescence-associated β-galactosidase (SA-β-gal) staining (×100) reveals more SA-β-gal-positive cells in MMD ECFCs than in normal ECFCs. F and G : In both MMD ECFCs and normal ECFCs, few caspase-3-positive cells are observed (×100). Scale bars : 100 μm. *p<0.05, **p<0.01, and ***p<0.001. ECFCs : endothelial colony-forming cells, MMD : Moyamoya disease. DAPI : 4',6-diamidino-2-phenylindole.

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  Fig. 2. Analysis of the major biological pathways of MMD ECFCs and verification of the associated genes. A : Functional gene enrichment analysis of identified differentially expressed genes (DEGs) as biological pathways. B : Validation of cell cycle-related genes by RT-qPCR. *p<0.05. ATM : ataxia telangiectasia mutated, ECFCs : endothelial colony-forming cells, MMD : Moyamoya disease, CDKN : cyclin-dependent kinase inhibitor, CCNA : cyclin A, CCNB : cyclin B, CCND : cyclin D, SIRT1 : sirtuin 1, RB1 : retinoblastoma, RT-qPCR : real-time quantitative polymerase chain reaction.

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  Fig. 3. Inhibition effect of CDKN2A in MMD ECFCs. A : CDKN2A-siRNA treatment effectively reduces CDKN2A mRNA expression by 70%. B : Cell viability analysis reveals increased cell growth of MMD ECFCs by knockdown of CDKN2A. C : Cell cycle analysis confirms inhibition of G1 arrest by knockdown of CDKN2A in MMD ECFCs. D and E : Senescence-associated β-galactosidase (SA-β-gal) assay (×100) shows a significantly decreased the cellular senescence of MMD ECFCs by knockdown of CDKN2A. F and G : Caspase-3 staining (×100) indicates that knockdown of CDKN2NA does not affect apoptosis in MMD ECFCs. H and I : Tube formation analysis demonstrates that knockdown of CDKN2A significantly improves the tubule formation capability of MMD ECFCs (×100). Scale bars : 100 μm. ***p<0.001. CDKN2A : cyclin-dependent kinase inhibitor 2A, NC: negative control, MMD : Moyamoya disease, ECFCs : endothelial colony-forming cells.

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  Fig. 4. Cellular senescence and cell cycle-related signaling pathways affected by inhibition of CDKN2A in MMD ECFCs. A : Representative western blot results indicate a significant increase in CDK4 and pRB by knockdown of CDKN2A in MMD ECFCs at 24 hours. On the other hand, CDKN1A, CDK2, and CDK6 are not affected by the knockdown of CDKN2A in MMD ECFCs. B : Densitometry graphs of the western blots are quantified, and the data are expressed as the mean±standard deviation. *p<0.05, ***p<0.001. NC : negative control, CDKN2A : cyclin-dependent kinase inhibitor 2A, pRB1 : phospho retinoblastoma1, MMD : Moyamoya disease, ECFCs : endothelial colony-forming cells.

Reference

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