Sneha Chebrolu

 Introduction. Growing evidence suggests ionizing radiation (IR) increases the risk of cardiovascular disease (CVD) in radiotherapy patients.^1,2 The effects of radiation damage are not usually observed until a decade after stopping treatment.¹ IR-induced endothelial senescence is a major mechanism for premature CVD in radiotherapy patients.^1,3 Senescence refers to aged cells that do not replicate but remain metabolically active.¹ They produce pro-inflammatory molecules such as IL-6 and ICAM1, which promotes atherosclerosis and other CVD.^1,3 Radiation therapy induces endothelial senescence by activating the NF-kB pathway and epigenetically upregulating CD44 expression.^4,5 Currently, the drugs used to manage IR-induced CVD are the same as for CVD of different etiologies.⁶ However, senolytic drugs have been identified as a way to specifically target and reduce viability of senescent cells.^7,8 Methods. Human umbilical vein endothelial cells (HUVEC) were used to examine the NF-kB pathway.⁴ HUVECs received radiation doses of 0-8Gy and were analyzed using MTT assay, Senescence-Associated β-Galactosidase (SA-βGal) assay, RT-qPCR, and immunofluorescence.⁴ CD44 activation was assessed using human coronary artery endothelial cells irradiated with 10Gy.⁵ Irradiated cells were stained for SA-βGal to confirm senescence while Western blot, immunofluorescence, and qPCR were used to measure CD44 levels. A quantitative monocyte adhesion assay was utilized to identify monocyte clusters.⁵ The senolytic drugs were tested in vitro using HUVEC cells and in vivo using murine animal models.⁸ Results. Nuclear p65 density was greater in irradiated cells and decreased in cells treated with PS1145 (NEMO inhibitor), proving IR activated the NF-kB pathway.⁴ Irradiated cells had senescent morphology, including larger cell size and abnormal nuclei, and quantitative analysis confirmed increased senescence levels.⁴ PCR analysis found increased mRNA levels of IL-6 in irradiated endothelial cells.⁴ Irradiation activated CD44 through demethylation at the CpG3 region.⁵ Anti-CD44 stain and Western blot showed irradiated cells had increased CD44 expression.⁵ The monocyte adhesion assay verified monocyte clusters have higher CD44 levels.⁵ Irradiated cells infected with short hairpin RNAs against CD44 had decreased monocyte adhesion, demonstrating CD44 is critical for adhesion.⁵ Both in vitro and in vivo testing of senolytic drugs reduced senescent cell viability but combination drug therapy of Quercetin and Dasatinib was more effective than monotherapy.⁸ Conclusions. Irradiation of endothelial cells induces premature senescence through activation of the NF-kB pathway and CD44 upregulation.^4,5 The resulting production of IL-6 and increased adhesion contribute to the accelerated CVD pathogenesis observed in radiotherapy patients.^1-5 Senolytic drugs preferentially reduce senescent cells and could be used to decrease IR-induced CVD.^7,8

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