Objective: Macrophage activation syndrome (MAS), a life-threatening inflammatory complication, is increasingly recognized in childhood-onset systemic lupus erythematosus (cSLE). It can be a challenge to differentiate active cSLE from MAS. We generated decision rules for discriminating MAS from active cSLE in newly diagnosed patients.
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Figure 3. Cerebral activations time-locked to sleep spindles and correlation between spindle-related activation and Reasoning abilities. (A) Activations time-locked to sleep spindles during NREM sleep. (B) Spatial correlation maps between activations time-locked to sleep spindles and Reasoning abilities. (C) Overlap between A (red) and B (green), with the conjunction of A and B shown in yellow. Statistical inferences were performed at a threshold of p < 0.001 (uncorrected) at the whole-brail level and p < 0.05, FWE corrected at the cluster level.
Figure 4. Semi-partial correlations between brain activations time-locked to spindles and Reasoning scores. ROI analyses revealed that Reasoning abilities were correlated with activations in the (A) the thalamus (partial correlation, r = 0.628, p < 0.001), (B) ACC/MCC (partial correlation, r = 0.585, p = 0.001), and (C) the bilateral putamen (partial correlation, r = 0.616, p = 0.001). Gender and the whole brain volume were included in the model as covariates of no interests. Values are standardized residuals (showing the partial correlation) and shown in standardized arbitrary units.
We have recently shown that induction of the p53 tumour suppressor protein by the small-molecule RITA (reactivation of p53 and induction of tumour cell apoptosis; 2,5-bis(5-hydroxymethyl-2-thienyl)furan) inhibits hypoxia-inducible factor-1α and vascular endothelial growth factor expression in vivo and induces p53-dependent tumour cell apoptosis in normoxia and hypoxia. Here, we demonstrate that RITA activates the canonical ataxia telangiectasia mutated/ataxia telangiectasia and Rad3-related DNA damage response pathway. Interestingly, phosphorylation of checkpoint kinase (CHK)-1 induced in response to RITA was influenced by p53 status. We found that induction of p53, phosphorylated CHK-1 and γH2AX proteins was significantly increased in S-phase. Furthermore, we found that RITA stalled replication fork elongation, prolonged S-phase progression and induced DNA damage in p53 positive cells. Although CHK-1 knockdown did not significantly affect p53-dependent DNA damage or apoptosis induced by RITA, it did block the ability for DNA integrity to be maintained during the immediate response to RITA. These data reveal the existence of a novel p53-dependent S-phase DNA maintenance checkpoint involving CHK-1.
To further explore the DNA damage response induced by RITA, we first assessed the phosphorylation status of p53 in response to RITA, as phosphorylation of p53 within the N-terminus is usually induced by genotoxic stress.19 p53+/+ HCT116 cells were treated with RITA over a concentration curve and assessed for Ser15, Ser20 and Ser46 phosphorylation of p53 (Figure 2a). We found that RITA induced N-terminal phosphorylation of p53 (Figure 2a). DNA damage is usually sensed by the PI-3K-related protein kinases ATM and ATR, which activate the transducer checkpoint kinases CHK-2 and CHK-1, respectively.20 ATM/ATR directly phosphorylates p53 at Ser15, whereas CHK-2/CHK-1 phosphorylates Ser20 on p53.21 Consistent with activation of the canonical ATM/ATR DNA damage response pathway, we found that RITA also induced phosphorylation of Ser345 on CHK-1 and Thr68 on CHK-2 (Figure 2a), and increased γH2AX in a dose-dependent manner (Figure 2a). These responses correlated with a dose-dependent increase of cells in sub-G1, indicative of apoptosis (Figure 2a, graph). In addition, we found that induction of phosphorylated CHK-1, CHK-2, p53 and γH2AX proteins was time-dependent (Figures 2b and c) and correlated with a time-dependent increase in cells in sub-G1 (Figure 2d). Thus, the DNA damage response induced by RITA is both dose- and time-dependent.
Here, we found that RITA activated the canonical ATM/ATR DNA damage response pathway that leads to activation of CHK-1 and CHK-2 phosphorylation. Intriguingly, however, and confirming our recent findings,17 we found that the induction of phosphorylated CHK-1 and γH2AX proteins observed in response to RITA was dependent on p53 status.
Previous studies have reported a p53-dependent DNA damage checkpoint.33, 34 Activation of a p53-dependent S-phase DNA damage checkpoint occurs to delay DNA synthesis and to allow time to resolve a potential replication block.33, 35 Our earlier immunohistochemical analyses showed that RITA induced a pan-nuclear localisation of γH2AX opposed to localisation to discrete nuclear foci.17 This type of DNA damage response indicates potential stalling of the replication fork or is mediated during processing of bulky DNA lesions.22 Indeed, we found that RITA induced a p53-dependent increase in replication fork number in a sub-population of replicons. Notably, a decline in replication fork rate is known to be consistent with increased rates of local origin activation and higher replication fork densities,25 indicating that RITA stalled DNA replication elongation and affected replication fork rate.
In conclusion, our study highlights a novel role for p53 in the activation of a p53-dependent S-phase replication checkpoint that involves CHK-1 and functions to protect the integrity of DNA. As we have previously shown that exposure of tumour cells to RITA leads to significant p53-dependent apoptosis in normoxia and hypoxia,15 it will be of particular interest to further examine the precise molecular mechanisms underlying this p53-dependent S-phase checkpoint in hypoxia.
Arteriovenous fistula (AVF) maturation is a process involving remodeling of venous arm of the AVFs. It is a challenge to balance adaptive AVF remodeling and neointima formation. In this study we temporally controlled Notch activation to promote AVF maturation while avoiding neointima formation.
Temporal Notch activation was controlled by regulating the expression of Notch transcription factor, RBP-Jκ, or dnMAML1 (dominant negative MAML2) in vascular smooth muscle cells (VSMCs). AVF mouse model was created and VSMC phenotype dynamic changes during AVF remodeling were determined.
Activated Notch was found in the nuclei of neointimal VSMCs in AVFs from uremic mice. We found that the VSMCs near the anastomosis became dedifferentiated and activated after AVF creation. These dedifferentiated VSMCs regained smooth muscle contractile markers later during AVF remodeling. However, global or VSMC-specific KO of RBP-Jκ at early stage (before or 1 week after AVF surgery) blocked VSMC differentiation and neointima formation in AVFs. These un-matured AVFs showed less intact endothelium and increased infiltration of inflammatory cells. Consequently, the VSMC fate in the neointima was completely shut down, leading to an un-arterialized AVF. In contrast, KO of RBP-Jκ at late stage (3 weeks after AVF surgery), it could not block neointima formation and vascular stenosis. Inhibition of Notch activation at week 1 or 2, could maintain VSMC contractile markers expression and facilitate AVF maturation.
This work uncovers the molecular and cellular events in each segment of AVF remodeling and found that neither sustained increasing nor blocking of Notch signaling improves AVF maturation. It highlights a novel strategy to improve AVF patency: temporally controlled Notch activation can achieve a balance between adaptive AVF remodeling and neointima formation to improve AVF maturation.
Adaptive vascular remodeling is required for AVF maturation. The balance of wall thickening of the vein and neointima formation in AVF determines the fate of AVF function. Sustained activation of Notch signaling in VSMCs promotes neointima formation, while deficiency of Notch signaling at early stage during AVF remodeling prevents VSMC accumulation and differentiation from forming a functional AVFs. These responses also delay EC regeneration and impair EC barrier function with increased inflammation leading to failed vascular remodeling of AVFs. Thus, a strategy to temporal regulate Notch activation will improve AVF maturation.
Notch and its ligands transduce signals between neighboring cells from signal-giving cells expressing Notch ligands to signal-receiving cells expressing Notch receptors. When a Notch ligand binds to Notch, the extracellular domain of the Notch receptor is cleaved, resulting in release of an intracellular Notch domain (abbreviated NICD). NICD can interact with RBP-Jκ, a primary transcription factor of Notch signaling . NICD displaces co-repressors from RBP-Jκ, and replaces them with co-activators [e.g., master mind 1 (MAML1)]. The binding of MAML1 to RBP-Jκ results in transcription of target genes, Hes1/5, Hey2, and VSMC markers, Myh11 and α-SMA [19,20,21,22,23]. A dominant negative MAML1 (dnMAML1) can block Notch activation. Therefore in this study we studied the roles of Notch transcription factor (RBP-Jκ and MAML1) on VSMC differentiation and neointima formation during AVF remodeling. We used temporal controlled expression of RBP-Jκ or dnMAML1 to manipulate Notch activation in VSMCs to improve AVF adaptive remodeling and maturation.
The above results demonstrate that Notch/RBP-Jκ activation is required for AVF maturation. Loss of Notch signaling blocks VSMC differentiation and migration and impairs AVF thickening. On the other hand, Notch/RBP-Jκ signaling is not required for maintenance of VSMC phenotype once the VSMC fate has been triggered/established. Thus, we propose that temporal regulation of Notch/RBP-Jк signaling may promote AVF maturation while preventing neointima hyperplasia. 153554b96e