Endothelial cell dysfunction and senescence are known to contribute to cardiovascular diseases [1]. These cells increase the expression and release of pro-inflammatory molecules and MMPs [4, 5], which have a causal relationship with cardiovascular diseases, including atherosclerosis, thrombosis, aneurysm pathophysiology, stroke, and heart infarct [10, 32]. In the current study, we used colchicine to ameliorate oxidative stress-induced dysfunction and senescence in endothelial cells.

Colchicine mitigated oxidative stress in endothelial cells treated with H2O2 (Fig. 1C), agreeing with the previously reported findings [25, 33, 34]. Oxidative stress can cause DNA damage, growth arrest, and premature cellular senescence [2]. Colchicine attenuated oxidative stress-induced DNA damage (Fig. 1A, D). Colchicine has been reported to reduce oxidative stress-induced DNA damage in ethanol-treated endothelial cells [5] and to provide anti-oxidative effects in endothelial cells and platelets [25, 34]. The oxidative stress and oxidative stress-induced DNA damage can activate NF-κB, MAPKs, and mTOR pathways and can cause cellular senescence [9, 20]. β-gal staining revealed that colchicine dampened senescence in endothelial cells treated with oxidative stress (Fig. 1B, E). Previous studies have shown the accumulation of senescent cells in atherosclerotic lesions [1, 8] and colchicine, by inhibiting cellular senescence (Fig. 1), can prevent the progression of atherosclerosis and promote atherosclerotic plaque stability [1, 8, 10]. Moreover, colchicine improved the relative protein expression of DNA repair protein KU80 (Fig. 1H; Table 1), aging marker Lamin B1 (Fig. 1I; Table 1), and attenuated the relative protein and mRNA expression of P21 (Fig. 1J, K; Tables 1 and 2) in oxidative stress treated endothelial cells. KU80 forms a heterodimer with KU70 and repairs double-strand DNA breaks through non-homologous end joining [35]. Reduced levels of KU80 have been observed in senescent cells [36] and impaired KU80 protein expression has been shown to cause telomere shortening [37], which can consequently result in cellular senescence. The aging marker Lamin B1 maintains nuclear stability and reduced Lamin B1 protein levels resulted in misregulated non-homologous end joining and homologous repair of DNA, leading to persistent DNA damage [38]. Lamin B1 protein level is decreased in senescent cells [39, 40] due to its reduced stability [39]. Loss of Lamin B1 protein expression can cause premature senescence [40]. P21 is an inhibitor of the cyclin-dependent kinase (CDK) and establishes indefinite growth arrest of senescent cells [2]. The induction of P21 protein expression led to senescence in human HT1080 fibrosarcoma cells [41]. Though colchicine rescued endothelial cells from oxidative stress-induced senescence and improved the expression of senescence markers in endothelial cells exposed to oxidative stress, however, colchicine alone increased the relative mRNA expression of P21 (Fig. 1K) and reduced the relative protein expression of KU80 (Fig. 1H) and Lamin B1 (Fig. 1I), suggesting its DNA-damaging and pro-senescent effects in untreated endothelial cells. It is worth noting that previously colchicine has been shown to induce senescence in lung cancer cells [30]. These findings suggest that colchicine inhibited oxidative stress-induced senescence (Fig. 1B, E) by improving KU80 (Fig. 1H) and Lamin B1 (Fig. 1I) protein expression and ameliorating P21 (Fig. 1J, K) expression at mRNA and protein levels.

The expression of senescence markers and DNA repair proteins is regulated by NF-κB, MAPKs, and mTOR pathways, and these pathways are known to contribute to cellular senescence [2, 9, 20]. Protein analysis showed that colchicine inhibited activation of NF-κB, MAPKs, and mTOR (Figs. 2, 3 and 4, and Table 1). NF-κB is known to promote inflammation and contribute to cellular senescence in vitro and in vivo [9, 10, 42]. NF-κB activation impairs KU80 protein expression, subsequently resulting in telomere shortening [43], which can lead to cellular senescence. In response to DNA damage, NF-κB increased the expression of P21 [44], leading to cell cycle arrest and senescence [2, 42]. This suggests that colchicine, via blocking NF-κB activation, can suppress P21 expression (Fig. 1J, K) and improve KU80 expression (Fig. 1H), resulting in reduced endothelial cell senescence (Fig. 1B, E). Moreover, NF-κB regulates SASP responses in dysfunctional and senescent endothelial cells [9, 10], where the transcriptional activity of NF-κB in senescent cells is regulated by MAPKs [20]. MAPKs also regulate the protein expression of cell cycle arrest proteins [20]. P38 increases the expression, stabilization, and promoter activity of P53, resulting in increased P21 protein expression [45, 46]. P38 increases the cytoplasmic accumulation of HuR via phosphorylating it [47]. HuR binds to P21 mRNA and increases its stability, consequently increasing P21 protein expression [47]. Blocking senescent signals via inhibiting P38 can mitigate Lamin B1 loss [39]. ERK also promotes the transcription of P21 [20]. Previous studies have shown that inhibiting P38 activation can subdue cellular senescence [48]. Taken together, it seems very likely that colchicine via inhibiting P38 (Fig. 3B) can improve Lamin B1 protein expression (Fig. 1I) and by blocking P38 (Fig. 3B) and ERK (Fig. 3C) pathways can subdue P21 mRNA and protein expression (Fig. 1J, K) in oxidative stress treated endothelial cells. Colchicine reduced the activation of the mTOR pathway in oxidative stress-induced endothelial cells (Fig. 4C). Still, when it was administered alone, it also increased the activation of the mTOR (Fig. 4C) pathway and its downstream molecule S6 (Fig. 4D), suggesting its pro-senescent role and adverse effects on autophagy. mTOR pathway is known to play a role in aging and senescence, and its inhibition increases longevity and delays senescence [21]. The activation of the mTOR pathway negatively regulates autophagy, resulting in accumulated damaged proteins and organelles, which can lead to the progression of cellular senescence [22, 49]. Blocking mTOR activation can improve mitochondrial function and reduce ROS levels [22], which can consequently provide protection against cellular senescence.

These findings indicate that colchicine inhibited oxidative stress-induced senescence (Fig. 1) via inhibiting the activation of NF-κB and MAPKs (Figs. 2 and 3; Table 1). Because the activation of these pathways in senescent cells regulates the expression of SASP factors [2, 9, 10, 20], therefore, we investigated the effect of colchicine on the regulation of mRNA expression of SASP factors in endothelial cells treated with oxidative stress. Agreeing with previously reported findings [5], our study showed that colchicine reduced the relative mRNA expression of SASP factors (Fig. 5). The expression of these SASP factors is regulated by P38 through the transcriptional activity of NF-κB [20]. The data suggest that via inhibiting the activation of NF-κB (Fig. 2) and P38 (Fig. 3), colchicine suppressed oxidative stress-induced mRNA expression of SASP factors (Fig. 5) in endothelial cells.

Senescent cells by increasing the expression of SASP factors (Fig. 5) [4, 5, 10], contribute to cardiovascular diseases via multiple mechanisms [10]. The enhanced expression and release of cytokines, chemokines, and cell adhesion proteins by these senescent endothelial cells causes tissue infiltration of monocytes, neutrophils, and platelets. The infiltration, accumulation, and activation of neutrophils, monocytes, and platelets contribute heavily to atherosclerosis and thrombosis [10]. Colchicine reduced the activation of monocytes [24] and hindered the adhesion of monocytes to endothelial cells by suppressing the expression of VCAM-1 and ICAM-1 [25]. In experimental animal studies, colchicine dampened the infiltration and recruitment of neutrophils and monocytes into the atherosclerotic plaques [23, 24] and infarct area of myocardium after myocardial ischemia [26]. Colchicine in vitro and in vivo ameliorated platelet activation and inhibited platelet-platelet, platelet-monocyte, and platelet-neutrophil aggregation [25, 33, 50] and thus can protect against atherosclerosis and thrombosis [25]. Moreover, colchicine in vitro mitigated the mRNA and protein expression of TNF-α, IL-1β, IL-6, IL-18, MCP-1, ICAM-1 and VCAM-1 (Fig. 5) [5, 25, 34] and in vivo ameliorated their mRNA expression, circulating levels and protein expression in experimental animal studies [23, 26, 27]. MCP-1 and IL-1β deficient mice and blocking MCP-1 in rats showed impaired cerebral aneurysm formation and progression [14, 15]. The lack of IL-1β, MCP-1, and the inhibition of MCP-1 receptor CCR2 decreased atherosclerotic formation [16,17,18]. From these findings, it can be postulated that colchicine, by inhibiting SASP factors (Fig. 5) [5,