Sepsis represents a global health concern, and patients with severe sepsis are at risk of experiencing MODS (multiple organ dysfunction syndrome), which is associated with elevated mortality rates and a poorer prognosis. The development of sepsis involves hyperactive inflammation, immune disorder, and disrupted microcirculation. It is crucial to identify targets within these processes to develop therapeutic interventions. One such potential target is Panx1 (pannexin-1), a widely expressed transmembrane protein that facilitates the passage of molecules smaller than 1 KDa, such as ATP. Accumulating evidence has implicated the involvement of Panx1 in sepsis-associated MODS. It attracts immune cells via the purinergic signaling pathway, mediates immune responses via the Panx1-IL-33 axis, promotes immune cell apoptosis, regulates blood flow by modulating VSMCs’ and vascular endothelial cells’ tension, and disrupts microcirculation by elevating endothelial permeability and promoting microthrombosis. At the level of organs, Panx1 contributes to inflammatory injury in multiple organs. Panx1 primarily exacerbates injury and hinders recovery, making it a potential target for sepsis-induced MODS. While no drugs have been developed explicitly against Panx1, some compounds that inhibit Panx1 hemichannels have been used extensively in experiments. However, given that Panx1’s role may vary during different phases of sepsis, more investigations are required before interventions against Panx1 can be applied in clinical. Overall, Panx1 may be a promising target for sepsis-induced MODS. Nevertheless, further research is needed to understand its complex role in different stages of sepsis fully and to develop suitable pharmaceutical interventions for clinical use. This global health concern accounts for approximately 50 million cases and 11 million deaths yearly . Those d+ current activates SFK (Src kinase), which phosphorylates Panx1, resulting in agonistic toxicity and impaired neuronal recovery in inflammatory conditions (The Panx (pannexin) protein family is found in vertebrates, first identified in 2000 . Panx annditions , 13. Addnditions . Elevatenditions .Studies have demonstrated Panx1’s role in inflammation , 17. At 2According to Sepsis-3 criteria, sepsis is characterized as a dysregulated host response to infection, wherein excessive inflammation and immunosuppression coincide; in cases where the body fails to effectively eliminate a high pathogen load, an exaggerated inflammatory response ensues, coupled with paradoxical anti-inflammatory regulation. The early phase of sepsis exhibits hyperinflammation involving multiple cell types and complex networks. While Panx1 is not directly engaged in the initial process, it plays a significant role by releasing signaling molecules like ATP, which can contribute to the proinflammatory response. Specifically, Panx1 promotes inflammasome activation  and faci2.1In sepsis, the delicate balance between protective and detrimental inflammation becomes crucial, as excessive inflammation can damage tissue. Inflammasome activation plays a vital role in hyperinflammation during sepsis, as it triggers the production and release of proinflammatory cytokines, such as IL-1β (Interleukin 1β) and IL-18 (Interleukin 18), and induces inflammatory cellular death, including pyroptosis and necroptosis. The inflammasome is a protein complex that responds to infection or damage. Cytoplasmic receptors like NLRP3  recruit ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) to activate caspase-1 . In cont7 receptors, which are ATP-gated ion channels. Although initially believed to be a K+ efflux channel , promoted by P2X7 receptors, activates NLRP3  , as well7 receptors , ultimately altering cytoskeletal structure, and enabling rapid DC migration , Panx1 collaborates with P2Xigration . Panx1 cchannels .6 receptors in response to CCL2-induced (C-C motif ligand 2) chemotaxis , the Panx1-IL-33 axis is distinct in enhancing bacterial elimination. Panx1-mediated purinergic signals activate P2X2 receptors, leading to IL-33 production in hepatocytes. Consequently, this axis recruits macrophages and neutrophils, effectively reducing MRSA infection (The Panx1-IL-33 (interleukin-33) axis is crucial in resolving excessive inflammation in the liver . Upon LPn sepsis . Notablynfection . These sMoreover, studies have suggested that Panx1 may also influence the population of infiltrating phagocytes during peritonitis development, further emphasizing its involvement in immunomodulation . As prev3.2In sepsis, the impairment of effector immune cells through various forms of programmed cell death exacerbates the immunosuppressive state. Pyroptosis, apoptosis, autophagy, and ferroptosis have been observed in immune cells during sepsis, collectively contributing to compromised immune function and hindering the body’s ability to combat infections . While t7 receptors  protein family, further contributing apoptosis , Panx1 is coupled to α1-adrenergic receptors and located on the caveolae of the cell membrane. Panx1 promotes VSMC contraction, following sympathetic adrenergic signals stimulation, resulting in vasoconstriction and increased blood pressure , 84. The2+ influx, facilitating pulmonary vasoconstriction and blood transfer from under-ventilated to well-ventilated areas during mechanical ventilation  by SFK. This activation leads to the opening of Panx1 hemichannels and subsequent Catilation . Previoutriction . However pathway . Further2 receptors, and TRPV4 are co-localized on caveolae, forming the Panx1-ATP-P2Y2R-PLC (phospholipase C)-PKCα (protein kinase C α)-TRPV4 pathway  pathway , 89. Stu pathway , 90. Thial cells .Panx1 may also impact NO production for NO-dependent regulation of vascular tone through inhibitory actions on eNOS . By modulating phosphorylation levels at Ser-1177 of eNOS, Panx1 can ultimately lead to reduced NO synthesis , 93. Add2+ wave propagation and relaxation of upstream VSMCs, ultimately resulting in vessel dilation  on platelets, which in turn promotes SFK phosphorylation. The interaction between SFK and Panx1 leads to the opening of Panx1 and the subsequent release of ATP. ATP then activates P2Xregation . Notablyregation .2+ flow, and promoting platelet aggregation, thereby contributing to arterial thrombosis . The integrity of the alveolar-capillary barrier is crucial for normal blood-air exchange, and any damage to this barrier can result in ALI or ARDS (acute respiratory distress syndrome). ALI can be triggered by inhaled pathogens, mechanical stress, and pro-inflammatory molecules . PulmonaDuring inflammation, pro-inflammatory molecules like TNF-α may activate Panx1 hemichannels in the lung, triggering the activation and recruitment of immune cells in the alveolar microcirculation. Simultaneously, mechanical stress arising from improper mechanical ventilation or autonomous respiratory attempts may activate Panx1 hemichannels in the alveolar microcirculation, leading to pyroptosis and further damaging the alveolar-capillary barrier, exacerbating pulmonary edema and promoting an inflammatory response. Although there is limited research on Panx1’s specific role in ALI, a recent study has suggested its potential involvement in pulmonary ischemia-reperfusion injury, pointing toward its potential therapeutic significance in treating ALI .5.2Immunodetection studies have provided insights into the distribution of Panx1 within the renal system, revealing its presence in various segments of renal tubules, including proximal tubules, thin descending limbs, and collecting ducts. Additionally, Panx1 expression is observed explicitly in VSMCs of afferent and efferent arterioles within the renal vasculature . This di2+ concentration . Panx1 contributes to brain dysfunction in sepsis-induced encephalopathy by promoting pyroptosis . The inhThese findings collectively highlight the significant role of Panx1 in CNS physiology and pathology, indicating its potential as a therapeutic target for managing neuroinflammatory conditions, specifically in sepsis. Further research is essential to fully comprehend the precise mechanisms and therapeutic implications of Panx1 modulation in the CNS.5.4Despite the growing evidence suggesting that Panx1 increases the inflammatory response, its specific role in the liver hasn’t been extensively discussed. However, recent studies have shed light on the significant involvement of Panx1 in liver physiology and pathology.Panx1 is highly expressed in both murine and human livers, under both healthy and diseased situations , and pri2R-IL-33 signaling axis. This leads to increased infiltration of Treg cells, thereby reducing bacterial infection-induced liver injury  and ALT . However, beyond the initial 24 hours, PANX1 deletion shows no significant protective effect, and by 48 hours, PANX1 knockdown fails to reduce the necrotic area . This su5.5- permeability of Panx1 determines its contribution to cardiac rhythm. When Ca2+ efflux from the sarcoplasmic reticulum activates Panx1 hemichannels on cardiomyocytes, it leads to high conductance. However, in the absence of Ca2+ current, episodic opening of Panx1 causes action potentials, potentially resulting in arrhythmias (Panx1 is widely expressed in the cardiovascular system and is crucial in cardiac rhythm regulation . The Cl-hythmias .7 receptors are involved in the release of cardioprotectants. Pre- or postconditioning with P2X7R agonists, such as ATP, has been shown to reduce the damaged area, indicating that the cardioprotective effects associated with Panx1 and P2X7 receptors are likely mediated through the release of ATP. However, the exact mechanism remains unknown (Panx1 serves distinct roles during acute and chronic heart inflammation, operating through separate signaling pathways. In cases of I/R injury, Panx1 and P2X unknown , 137.6 receptors, activates the heterotrimeric G12 family G protein. The activation promotes the expression of fibrogenic genes, leading to myocardial fibrosis (However, in chronic inflammation, such as myocardial fibrosis, ATP via Panx1, combined with P2Xfibrosis . The rol610panx1 have shown promise and potential for future therapeutic approaches (There are currently no drugs available that specifically target and inhibit Panx1. Still, some compounds have been reported to exhibit inhibitory effects on Panx1 activity and have been extensively studied in experimental settings. Among these compounds, probenecid, carbenoxolone, and proaches , 140. Deproaches .7 receptors, which can limit its application in experiments (Probenecid, a medication commonly used for gout, is clinically used to increase effective concentrations of antibiotics, chemotherapeutics, and other drugs . Probeneeriments . Nevertheriments , skeletaeriments , as welleriments . Intereseriments , 150. AlInitially developed as an anti-ulcer medication, Carbenoxolone inhibits Panx1 activity by binding to its first extracellular loop of Panx1 . It has 10panx1, a 10-amino acid peptide derived from the extracellular loop of the Panx1 protein, provides selective inhibition of Panx1 without affecting ATP-evoked currents, distinguishing it from probenecid and carbenoxolone (10panx1 effectively inhibits Panx1-mediated ATP release and inflammasome activation, reducing inflammatory responses in various experimental models (10panx1 as a therapeutic agent for sepsis are still being investigated, as research by Chen et al. has suggested that 10panx1 may exacerbate sepsis-induced animal lethality (noxolone . Studiesl models –162. Howethality .Though inhibitors of Panx1 show promising outcomes in animal models of sepsis, there is still a long way before clinical application in treating sepsis, as no clinical trials have been reported yet. Moreover, as discussed above, Panx1 involves both hyperinflammation and immunosuppression. As a result, solely targeting it may not be sufficient to address the complexity of sepsis and sepsis-induced MODS. It could worsen immune disorders and organ damage in patients. Therefore, further evidence is required to understand the pattern of Panx1 hemichannel activation in sepsis before exploring its clinical therapeutic potential. More comprehensive research is needed to elucidate Panx1’s role and potential as a therapeutic target in sepsis and sepsis-induced MODS.7This comprehensive review delves into the potential roles of Panx1 in the pathogenesis of sepsis, examining its contribution to the excessive inflammatory response in the early phase, immunosuppression in the late phase, and impairment of microcirculation. Considering Panx1’s expression in multiple organs and its involvement in organ inflammatory injury, it is reasonable to suggest that Panx1 may play a role in the development of MODS. Inhibiting Panx1 hemichannels emerges as a promising therapeutic strategy against sepsis and sepsis-associated MODS; however, further research is essential before its clinical translation can be realized. As the scientific investigation on Panx1 hemichannels in sepsis is limited, it becomes crucial to explore its distinct functions at different phases of sepsis and understand how it contributes to organ injuries. This makes Panx1 an intriguing potential therapeutic target for sepsis and sepsis-induced MODS, offering a fresh approach to therapeutic interventions against these challenging disorders. Given its significant involvement in various critical processes, Panx1 represents a promising therapeutic target for sepsis and MODS.XC wrote the paper, SY proposed the theme of article, LM assisted in writing, and YL and HH provided guidance. All authors contributed to the article and approved the submitted version.


Notably, genes associated with the myostatin (9 genes) and mammalian target of rapamycin complex 1 (mTORC1) (9 genes) signaling pathways were most represented. Compared to mTORC1 signaling mRNAs, more MSTN signaling-related mRNAs (7 of 9) were altered post-exercise, regardless of the bout, and RHEB was the only mTORC1-associated mRNA that was upregulated following exercise. Phosphorylated (phospho-) p70S6K (Thr389)  and follistatin protein levels  increased post-exercise, regardless of the bout, whereas phospho-AKT (Thr389), phospho-mTOR (Ser2448), and myostatin protein levels remained unaltered. These data continue to suggest that performing resistance exercise to volitional fatigue, regardless of load selection, elicits similar transient mRNA and signaling responses in skeletal muscle. Moreover, these data provide further evidence that the transcriptional regulation of myostatin signaling is an involved mechanism in response to resistance exercise.Although transcriptome profiling has been used in several resistance training studies, the associated analytical approaches seldom provide in-depth information on individual genes linked to skeletal muscle hypertrophy. Therefore, a secondary analysis was performed herein on a muscle transcriptomic dataset we previously published involving trained college-aged men (n = 11) performing two resistance exercise bouts in a randomized and crossover fashion. The lower-load bout (30 Fail) consisted of 8 sets of lower body exercises to volitional fatigue using 30% one-repetition maximum (1 RM) loads, whereas the higher-load bout (80 Fail) consisted of the same exercises using 80% 1 RM loads. Vastus lateralis muscle biopsies were collected prior to (PRE), 3 h, and 6 h after each exercise bout, and 58 genes associated with skeletal muscle hypertrophy were manually interrogated from our prior microarray data. Select targets were further interrogated for associated protein expression and phosphorylation induced-signaling events. Although none of the 58 gene targets demonstrated significant bout x time interactions, ~57% (32 genes) showed a significant main effect of time from PRE to 3 h (15↑ and 17↓,  Resistance training promotes increases in strength and skeletal muscle hypertrophy . IncreasSkeletal muscle molecular responses to resistance exercise are often determined by examining changes in DNA methylation ,12,13,14Cell Adhesion” , “Cell Cycle” , “Cell Death” , and “Inflammation” . Accordingly, human resistance exercise research utilizing bioinformatics to interpret acute or longer-term skeletal muscle transcriptomic datasets have generally garnered ambiguous information to the detriment of “drowning out” genes or gene pathways that may be critical to skeletal muscle hypertrophy. This is evident with our own findings suggesting that 30 Fail and 80 Fail exercise bouts acutely altered muscle mRNAs predicted to affect “inflammatory signaling”, “apoptosis signaling”, and “gonadotropin-releasing hormone signaling” [As it relates to the interrogation of genes mechanistically associated with skeletal muscle hypertrophy, existing differential expression pipelines and subsequent pathway analysis tools are limited in their ability to predict a gene’s role in this process. Likewise, gene lists for skeletal muscle hypertrophy  are perhaps misleading given that genetic manipulation models and other mechanistic preclinical models  have indicated that the knockout, knockdown, or overexpression of dozens of genes alter skeletal muscle mass in adult rodents ,30,31,32gnaling” . It has gnaling” ,35,36. Tgnaling” . As a reHerein, we sought to fulfill multiple aims in the current study. First, we examined how 30 Fail versus 80 Fail training affected skeletal muscle mRNAs identified from a literature search that have been linked to skeletal muscle mass regulation using preclinical models ,25,26. Fwww.ncbi.nlm.nih.gov/geo/; GEO accession numbers: GSE220928 for DNA methylation data (public on 14 December 2022), and GSE220899 for mRNA array data (scheduled for release on 1 June 2023)).This study was conducted with prior review and approval from the Auburn University Institutional Review Board and in accordance with the most recent revisions of the Declaration of Helsinki . Raw DNA methylation data and mRNA data can be found in the Gene Expression Omnibus  were recruited from the local community, and all were required to have participated in lower-body training at least once per week over the last 6 months. Other details regarding inclusion and exclusion criteria can be found in the parent publication by Sexton et al. .A more comprehensive description of the study design and methodology can be found in Sexton et al. . BrieflyMuscle tissue processing for simultaneous DNA, RNA, and protein isolation. In-depth details regarding tissue processing can be found by Sexton et al. [n et al. . BrieflymRNA and DNA methylation arrays. Isolated RNA was shipped to a commercial laboratory for transcriptomic analysis . Following the quantification of gene expression using the Clariom S Assay_Human mRNA array. Raw.CEL files were uploaded into the Transcriptome Analysis Console v4.0.2 (TAC)  and subsequently annotated via the h. sapiens genome (Hg 38 build). Data were then normalized using the robust multiarray average (RMA) normalization method and are presented as log2-signal intensity. Isolated DNA was shipped to a commercial laboratory , and methylation analysis was performed using the Infinium MethylationEpic BeadChip Array  per the manufacturer’s instructions. Additional details regarding data analysis can be found in Sexton et al. . These dWestern blotting. Protein samples that were prepared as discussed above were loaded onto 4–15% SDS-polyacrylamide gels (Bio-Rad) and subjected to 50 min of electrophoresis (180 V) using SDS-PAGE running buffer (VWR). Proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad) using a wet blotting apparatus at 200 mA for 2 h. Following transfers, PVDF membranes were Ponceau stained and imaged in a gel documentation system  to ensure equal protein loading between lanes. Following 1 h blocking at room temperature with 5% nonfat milk powder in Tris-buffered saline with 0.1% Tween-20 , membranes were incubated overnight (pan proteins) or for ~48 h (phospho-proteins) with the following antibodies at a 1:1000 dilution in TBST: (i) rabbit anti-human phospho-mTOR , (ii) rabbit anti-human pan mTOR , (iii) rabbit anti-human p-p70S6K , (iv) rabbit anti-human pan p70S6K , (v) rabbit anti-human phospho-AKT , (vi) rabbit anti-human pan AKT , (vii) mouse anti-human follistatin , and (viii) mouse anti-human myostatin . Following primary antibody incubation periods, membranes were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies  in TBST with 5% BSA at room temperature for 1 h. An enhanced chemiluminescent reagent  was used to develop membranes in a dark gel documentation station , and band densitometry was performed using associated software. Densitometry values of FST and MSTN were normalized to Ponceau densities that corresponded to ~25–75 kD. Phosphorylated target band densities were divided by the pan densities of respective targets to obtain a ratio. All data are expressed as relative expression units.Literature review to identify candidate genes mechanistically associated with skeletal muscle hypertrophy. Our strategy for identifying candidate genes mechanistically associated with skeletal muscle hypertrophy was three-fold. First, the five genes provided by GOC (GO: 0014734) were included in our gene target list . Next, a systematic literature review published in 2018 by Verbrugge and colleagues provided a list of 47 additional genes from mechanistic preclinical studies in which the knockout, knockdown, or overexpression of these genes were found to increase skeletal muscle weight or myofiber cross-sectional area [UBR5 [RPTOR [TRIM28 [MAPK8/JNK1 [PSMC4 [ATG7 [YAP1 [TPT1, GNAS, PPARGC1A, CAST, ASB15, MCU, BMPR1A, NR3C1, THRA, SMAD4, KLF10, DGAT1, RHEB, NR4A1, IGF1, NCOR1, MSTN, MAPK8, PSMC4, YAP1, IKBKB, UBR5, NOL3, GRB10, TRIM28, EIF2B5, PPARD, XIAP, ATG7, AKT1, AGTR1, DGKZ, LTBP4, PLD1, ACVR2B, RPTOR, ESR1, TP53INP2, CRTC2, ADRB2, JUNB, INHBB, SKI, INHBA, FST, GPRASP1, FSTL3, MMP9, CAMKK1, PAPPA, CRYM, BDKRB2, UCN3, AR, IGFBP5, MTOR, MYMK, and MYOC.nal area . Finallyea [UBR5 , RPTOR [5 [RPTOR , TRIM28 PK8/JNK1 , PSMC4 [1 [PSMC4 , ATG7 [3C4 [ATG7 , and YAPG7 [YAP1 . The finp < 0.01 and if the expression at PRE did not differ between conditions . All Western blot data were analyzed with SPSS  using two-way within-within repeated measures ANOVAs, and LSD post hoc tests were used to decompose the main effects of time. All data in tables and figures are presented as mean ± standard deviation (SD) values, and, in all cases except methylation data, individual respondent data are also presented. As described by Sexton et al.  Transcrip < 0.01), and ~26% showed a main effect of time from PRE to 6 h . ACVR2B, BMPR1A, FST, FSTL3, INHBA, INHBB, MSTN, SKI, SMAD4) made up a large portion of the 58 genes interrogated. Notably, 7 of these 9 genes  displayed a main effect of time. Given that several of these mRNAs were dynamically altered, we had access to the methylation data of these genes from Sexton et al. [Genes involved with MSTN signaling  did not show significant alterations in methylation status at 3 h or 6 h, indicating that the upregulation in FST was likely unrelated to DNA methylation events. Alternatively, of the 160 SKI-associated CpG sites investigated, several (77 during 30 Fail and 67 during 80 Fail) showed significant paradoxical methylation at 3 h, indicating that the upregulation in SKI was also unlikely unrelated to DNA methylation events. also made up a large portion of the 58 genes interrogated. Of these genes, RHEB  was the only one that was upregulated. Genes involved with mTORC1 signaling that were downregulated included IGFBP5  and DGKZ . Nine genes that are part of the mTORC1 signaling cascade p = 0.001; Given that gene targets involved with MSTN and mTORC1 signaling were highly prevalent on our list of 58 genes, we opted to perform Western blotting on these targets. AKT-mTORC1 pathway proteins are presented in p < 0.001; Protein abundance data for follistatin and myostatin proteins are presented in This investigation aimed to extend the findings reported by Sexton et al.  by deterACVR2B, BMPR1A, FST, FSTL3, INHBA, INHBB, MSTN, SKI, SMAD4) [MSTN has been demonstrated as having a role in the suppression of the skeletal muscle growth [ACVR2B gene encodes for a receptor that mediates MSTN signaling through phosphorylation  of SMAD transcription factors [FST is essential for muscle fiber formation and growth by inhibiting the myostatin binding [FSTL3 similarly inhibits myostatin and activin A, which suppresses their functionality and promotes muscle growth [INHBA is a muscle growth inhibitor and encodes for activins which are a part of the TGF-β superfamily and have roles in the development regulation [INHBB is also a part of the TGF-β superfamily and is a muscle growth inhibitor [SKI also impairs MSTN signaling due to its ability to inhibit the activity of the SMADs [SMAD4 acts as an intracellular mediator of TGF-β signaling [MSTN and ACVR2B), whereas there was an up-regulation in mRNA targets that inhibit MSTN pathway activation . These findings exhibit similarities and differences with past reports in the literature. For instance, multiple studies have reported that skeletal muscle MSTN mRNA is downregulated in response to acute and longer-term resistance training periods [ACVR2B mRNA expression in older adults (age = 68 ± 6 years) is not altered, while another study has reported increased ACVR2B mRNA expression in younger recreational athletes (age = 27 ± 5 years) rehabilitating from ACL surgery [FST mRNA expression has been shown not to be affected in skeletal muscle following one and multiple sessions of resistance exercise [MSTN mRNA levels, this was likely due to biopsy sampling being too early to detect a downregulation in protein levels. In this regard, others have shown that immediate post-exercise alterations in PGC1-α mRNA levels lead to protein expression changes that are measurable ~24 h following the exercise bout [Among the genes identified during our literature review, as well as those identified by Verbrugge et al. , nine of, SMAD4) . MSTN hae growth , possible growth . The ACV factors . FST is  binding . FSTL3 se growth ,44. INHBgulation ,46. INHBnhibitor ,48. SKI he SMADs ,50, and ignaling . Seven o periods ,53,54,55 surgery ,57, and exercise ,59. Indeexercise , more reise bout . Thus, aAKT1, DGKΖ, EIF2B5, IGF1, IGFBP5, MTOR, PLD1, RHEB, RPTOR) were also included in the current analysis. Briefly, the mTORC1 pathway is considered an anabolic signaling hub, and its activation in skeletal muscle involves an upregulation of several signaling proteins by way of phosphorylation which leads to increased translation initiation resulting in increased muscle protein synthesis [AKT1 encodes a kinase that acts to activate the mTORC1 protein kinase [DGKΖ encodes for an enzyme that acts to increase cellular phosphatidic acid levels in response to mechanical overload, and phosphatidic acid binds to mTOR, resulting in mTOR activation [IGFBP5 is a binding protein that transports insulin-like growth factor variants in circulation and modulates ligand effectiveness [EIF2B5 encodes for a translation initiation factor which acts to increase rates of translation initiation and, subsequently, protein synthesis [IGF1 encodes for a growth factor that supports muscle growth by activating the tyrosine kinase IGF1 receptor, thus stimulating PI3K-AKT signaling [RPTOR encodes a protein that binds to and upregulates the mTORC1 signaling [RHEB encodes for a GTPase that stimulates the mTORC1 activity [RHEB mRNA increased during the post-exercise period, DGKZ, IGF1, and IGFBP5 mRNAs were downregulated, and no changes were observed for AKT1, EIF2B5, MTOR, PLD1, or RPTOR. These findings both agree and disagree with prior findings in the literature. For instance, resistance exercise has been reported to transiently up-regulate IGF1 mRNA in the post-exercise period [RHEB findings agree with Wang et al. [IGFBP5 mRNA data agree with a report by Dennis et al. [MTOR mRNA findings also align with data published by Drummond et al. [MTOR mRNA was not affected 3 h following resistance exercise. When considering the collective evidence, it appears that certain genes critical to mTORC1 signaling  may not be transcriptionally responsive to resistance exercise, whereas RHEB is upregulated, and this may be a transcriptional mechanism involved with the hypertrophic response to resistance exercise.Nine genes involved with mTORC1 signaling  were interrogated, given the robust changes we observed in MSTN-related mRNAs in response to both modes of resistance exercise. The interrogation of these targets with the current study design is novel given that they were not investigated in the 30 Fail versus 80 Fail Mitchell et al.  or Haun Pathway coverage of genes mechanistically associated with skeletal muscle hypertrophy is underrepresented in traditional pipelines used to elucidate differential gene expression. When considering the collected data, it is apparent that a bout of lower and higher load training to failure similarly affects the 58 interrogated mRNAs mechanistically associated with skeletal muscle hypertrophy. Further, select mTORC1 signaling markers are similarly affected between bouts, as is the protein expression of follistatin. Critically, our secondary analysis provides in-depth information regarding how the mRNA expression of genes mechanistically associated with skeletal muscle hypertrophy was affected following two unique resistance exercise bouts to failure. Regarding investigations seeking to elucidate underlying mechanisms of muscle growth, we recommend that researchers adopt a targeted approach with directed attention to gene expression analysis.


Trachinotus ovatus liver was observed against high-temperature stress. Through differential expression and short time-series expression miner (STEM) analyses, some high-temperature-related genes and biological pathways were screened, which were mainly related to protein balance, hypoxia adaptation, and energy metabolism. Our results suggest that protein dynamic balance and function, hypoxia adaptation, and energy metabolism transformation are crucial in response to acute high-temperature stress. These results contribute to understanding the molecular response mechanism of T. ovatus under acute heat stress and provide novel insights into the selection and breeding of heat-tolerant cultivars and the high-quality development of aquaculture.In this study, the transcriptomic response of Trachinotus ovatus is a major economically important cultured marine fish in the South China Sea. However, extreme weather and increased culture density result in uncontrollable problems, such as increases in water temperature and a decline in dissolved oxygen (DO), hindering the high-quality development of aquaculture. In this study, liver transcriptional profiles of T. ovatus were investigated under acute high-temperature stress (31 °C and 34 °C) and normal water temperature (27 °C) using RNA sequencing (RNA-Seq) technology. Differential expression analysis and STEM analysis showed that 1347 differentially expressed genes (DEGs) and four significant profiles  were screened, respectively. Of these DEGs, some genes involved in heat shock protein (HSPs), hypoxic adaptation, and glycolysis were up-regulated, while some genes involved in the ubiquitin-proteasome system (UPS) and fatty acid metabolism were down-regulated. Our results suggest that protein dynamic balance and function, hypoxia adaptation, and energy metabolism transformation are crucial in response to acute high-temperature stress. Our findings contribute to understanding the molecular response mechanism of T. ovatus under acute heat stress, which may provide some reference for studying the molecular mechanisms of other fish in response to heat stress.  Aquaculture is one of the fastest-growing sectors of the food industry worldwide and provides about 50% of animal protein for humans , which iAcipenser schrenckii) [Salmo salar) [Cyprinus carpio communis) [Paralichthys olivaceus) and turbot  [Notothenia rossii) [Cyprinidae) [Acipenser dabryanus) [Oreochromis niloticus) [Oncorhynchus mykiss) [S. salar [Oryzias latipes) [Channa punctata) [Salvelinus fontinalis) [Fish response to heat stress is highly complex. Heat stress can cause adverse effects on fish behavior, such as reduced feeding in amur sturgeon (renckii)  and Atlao salar)  and disoommunis) . Heat stmaximus) , marbled rossii) , gymnocyrinidae) , and Yanbryanus) , inhibitloticus)  and rain mykiss) , suppresS. salar , and exalatipes) . In addiunctata)  and brootinalis)  with theOncorhynchus tshawytscha) [Ctenopharyngodon idella) [A. dabryanus [Transcriptome RNA sequencing (RNA-Seq) technology has been used to understand the molecular mechanisms of fish response to heat stress, and some examples are as follows. Under heat stress, some genes related to protein folding and degradation, oxidative stress, immune response, and cell signal transduction were up-regulated in the gill tissue of chinook salmon (wytscha) . Some ge idella) . Some HSabryanus . These sabryanus ,23,24,25T. ovatus), belonging to the genus Trachinotus, is one of the most important species of warm-water fish and is widely distributed in coastal areas of Fujian, Guangdong, Guangxi, and Hainan provinces of China [T. ovatus is 22–28 °C [T. ovatus and threatens the healthy and sustainable development of aquaculture [T. ovatus have mainly focused on disease control [T. ovatus has also been discussed, which mainly focused on larvae and juveniles [T. ovatus. The liver plays a central role in fish carbohydrate, lipid, and protein metabolism and is one of the key tissues affected by high-temperature conditions [T. ovatus liver tissue in high-temperature environments using transcriptomics and other omic technologies, which helps understand the molecular response mechanism of this species.Golden pompano  analysis. Combined with the published research results, the main biological processes and their molecular mechanisms were clarified in response to acute heat stress in T. ovatus. The results of our study have importance for the selective breeding of high-temperature tolerance varieties in T. ovatus, the regulation of temperature and oxygen in aquaculture water, the selection of aquaculture waters, and the improvement of risk resistance in cultured fish. It can also provide a reference for the study of responding to the molecular mechanism of heat stress in other fish.In this study, RNA-Seq was used to study the changes in transcription levels in the liver tissue of T. ovatus were purchased from Zhanjiang Hist Aquatic Technology Co., Ltd., Zhanjiang, China, and their average standard lengths (SL) and weights (SW) were 21.1 ± 1.24 cm (mean ± SD (standard deviation)) and 333.8 ± 61.21 g, respectively. After transporting to the experimental base, T. ovatus were evenly placed into three plastic tanks (1.0 m × 1.0 m × 1.5 m) with 30 experimental fish per tank (T. ovatus were domesticated for two weeks to minimize stress response. The domestication conditions were as follows: temperature (27.0 ± 0.5 °C), salinity (24 ± 1‰), and DO (6.0 ± 0.3 mg/L). During the domestication period, T. ovatus were fed twice a day with commercial pelleted feed, and 30–40% of the seawater in each tank was regularly changed every day.A total of 90 healthy and disease-free per tank . Before HT31), and 34 °C , were set up in each parallel group. Before the heat stress experiment, all experimental fish were fasted for 24 h and were not fed during the experiment. After the beginning of the experiment, water temperature in three parallel groups was gradually increased from 27 °C to 34 °C at 1 °C/h and maintained at 34 °C for 2 h using heating rods (WN-B15). Three fish were selected randomly from each parallel group at 27 °C, 31 °C, and 34 °C (kept for 2 h) and anesthetized with phenolic. After measuring body length and weight, fish were dissected, and their liver tissues were removed and put into liquid nitrogen for cryopreservation. Nine liver samples were collected at each temperature . A total of 27 samples were collected at three temperature groups .Three parallel groups were set up in the experiment, and three temperature gradients, including 27 °C , 31 °C  at three temperature groups were used for all experiments and subsequent analyses. According to the manufacturer’s instructions, total RNA was extracted from nine liver samples using a Trizol reagent . Agilent 2100 Bioanalyzer  and RNase-free agarose gel electrophoresis were used to detect the integrity of total RNA and the presence of contamination. Qubit 2.0 Fluorometer (Invitrogen) was used to quantify RNA concentration accurately, and a NanoPhotometer spectrophotometer  was used to detect the purity of total RNA. Only RNA samples with the required integrity, concentration, and purity were used for subsequent library construction and Illumina RNA-Seq.The messenger RNAs (mRNAs) with poly (A) tails were enriched from qualified total RNA using magnetic beads with oligo (dT), and the enriched mRNA was fragmented into short fragments by ultrasound. The short mRNA fragments were reverse-transcribed into first-strand complementary DNA (cDNA) using random primers, and M-MuLV reverse transcriptase, followed by second-strand cDNA synthesis using RNase H and DNA polymerase I. The cDNA fragments were purified with a QiaQuick PCR extraction kit , end-repaired, poly (A) added, and ligated to Illumina sequencing adapters. The cDNA of about 200 bp was screened using AMPure XP beads. PCR amplification was then performed, and the PCR product was purified using AMPure XP beads to obtain a cDNA library. To obtain high-quality sequencing data, the quality of the cDNA library was strictly checked using the same method described for total RNA. The qualified cDNA library was sequenced using Illumina HiSeqTM 4000 by Gene Denovo Biotechnology Co. .To ensure the quality of data and downstream analyses, the raw reads obtained from sequencing were filtered by fastp version 0.18.0  to removT. ovatus was downloaded from Figshare [T. ovatus by HISAT 2.2.4 [The gene annotation file of the reference genome and gene model of Figshare , and theAT 2.2.4 , obtainip < 0.05 were considered significantly enriched.The mapped reads were normalized as fragments per kilobase of transcript per million mapped reads (FPKM), and further data analyses were based on normalized data. Pearson’s correlation coefficient was calculated to evaluate the repetition correlation between parallel samples within every temperature group. Differentially expressed gene (DEG) analysis was performed using DESeq2 , and thep < 0.05. GO term and KEGG pathway enrichment analyses were performed on each significant gene expression profile, understanding the specific biological processes in response to heat stress.Based on the transformed FPKM values, all the genes having similar expression patterns were classified by STEM . The parT. ovatus, protein–protein interaction (PPI) network analysis of DEGs in each significant profile was performed using the String v10 [Based on the protein interaction data from ring v10  databasering v10 . Finally® Uni All-in-One First-Strand cDNA Synthesis SuperMix for qPCR Kit  following the manufacturer’s instructions. For confirming the reliability of transcriptome data, 11 DEGs were randomly selected for qRT-PCR validation, and the β-actin gene was used as an internal reference gene. The gene-specific primers were designed using Primer-BLAST in National Center for Biotechnology Information (NCBI) based on the target gene sequences . Total R® Green qPCR Super Mix , 1 μL cDNA, 0.4 μL forward primer (10 mmol), and 0.4 μL reverse primer (10 mmol). Light Cyeler 96  was applied for qRT-PCR, and the PCR reaction cycle program was as follows: pre-denaturation at 94 °C for 30 s, followed by 40 cycles of 94 °C for 5 s and 60 °C for 30 s. Each sample was prepared in three technical replicates, and each experiment contained a negative control without template DNA. After qRT-PCR amplification, the amplification curve and melting curve were examined to evaluate specific amplification and the relative expression levels of target genes were calculated by the 2−△△CT method. The data of 11 selected genes were compared and obtained from qRT-PCR and transcriptome sequencing to validate the reliability of transcriptome sequencing data.The final volume of qRT-PCR reaction was 20 μL, including 8.2 μL RNase-free water, 10 μL 2× Perfect StartT. ovatus, and the matching degree of nine samples was 77.72–94.03% with an average matching rate of 90.98% .A total of 55 level 2 GO terms were obtained by GO enrichment analysis of 1347 DEGs, of which 24 belonged to biological processes (BP), 12 belonged to molecular functions (MF), and 19 belonged to cellular components (CC). Separate GO enrichment analysis of each comparison group  showed tp < 0.05). The significantly enriched GO terms included angiogenesis, antigen processing and presentation of peptide antigen via MHC class I and apoptotic process, carbohydrate metabolic process, cellular response to stress, immune system process, misfolded or incompletely synthesized protein catabolic process, regulation of protein folding, response to oxidative stress, tRNA metabolic process, proteasome-mediated ubiquitin-dependent protein catabolic process, regulation of ubiquitin-protein transferase activity, and so on.Level 3 GO enrichment analysis showed  that a tp < 0.05) in three comparison groups were classified into six major categories. Metabolism, genetic information processing, and organismal systems were common in three comparison groups. Cellular processes and human diseases were common to CT vs. HT31, and HT31 vs. HT34, and environmental information processing was unique to CT vs. HT31. Environmental information processing processes, such as signaling, were primarily activated when water temperature rose from 27 to 31 °C, further activating more physiological processes in the liver of T. ovatus.The big categories of KEGG pathway enrichment analysis of DEGs A–C showep < 0.05). The significantly enriched pathways included biosynthesis of amino acids, one carbon pool by folate, protein processing in the endoplasmic reticulum, glycolysis/gluconeogenesis, carbon metabolism, IL-17 signaling pathway, HIF-1 signaling pathway, proteasome, peroxisome, protein export, and so on. These significantly enriched pathways were related to carbohydrate metabolism, amino acid metabolism, environmental adaptation, immune system, translation, transcription, folding, sorting, and degradation.In the subclass KEGG enrichment analysis D–F, 231,p = 4.5 × 10−34, 4.0 × 10−31, 1.4 × 10−10, and 2.6 × 10−8). Profiles 4, 3, 0, and 7 had 315, 342, 180, and 170 genes, respectively, which were significantly higher than those of the other four non-significant profiles (49–108 genes). Four significant expression profiles showed different expression trends . The significantly enriched GO terms of DEGs in profile four mainly included carbohydrate catabolic process, angiogenesis, cellular carbohydrate metabolic process, single-organism carbohydrate metabolic process, regulation of carbohydrate metabolic process, regulation of cellular carbohydrate metabolic process, and so on. The significantly enriched GO terms for DEGs in profile 3 included antigen processing and presentation of exogenous peptide antigen via MHC class I, positive regulation of ubiquitin-protein transferase activity, positive regulation of protein ubiquitination, proteasome complex, proteasome-mediated ubiquitin-dependent protein catabolic process, and so on. The predominant enriched GO terms of DEGs in profile 0 included RNA binding, arginine N-methyltransferase activity, rRNA methyltransferase activity, tRNA methyltransferase activity, ubiquitin-like protein transferase activity, ubiquitin-protein transferase activity, and so on. The significantly enriched GO terms for DEGs in Profile 7 mainly include sperm–egg recognition, protein folding, ‘de novo’ protein folding, small molecule binding, regulation of ATPase activity, complex protein assembly, etc.GO enrichment analysis at level 3  showed tp < 0.05). The significantly enriched KEGG pathways in profile 4 included primarily amino acid metabolism, cancer, signal transduction, carbohydrate metabolism, and other related pathways, which were refined to the biosynthesis of amino acids, phenylalanine metabolism, microRNAs (miRNAs) in cancer, HIF-1 signaling pathway, glycolysis/gluconeogenesis, and so on. The significantly enriched KEGG pathways in profile 3 included folding, sorting and degradation, translation, carbohydrate metabolism, and other related pathways, and more specific pathways included proteasome, protein export, ribosome biogenesis in eukaryotes, and so on. The significantly enriched KEGG pathways in profile 0 included primarily translation, transcription, folding, sorting and degradation, and other related pathways, which were refined to ribosome biogenesis in eukaryotes, spliceosome, ubiquitin-mediated proteolysis, and so on. The significantly enriched KEGG pathway in profile 7 included carbohydrate metabolism, amino acid metabolism, immune system, folding, sorting and degradation, and other related pathways, which could be refined to more specific pathways, such as glycolysis/gluconeogenesis, citrate cycle (TCA cycle), cysteine and methionine metabolism, antigen processing and presentation, protein processing in the endoplasmic reticulum, and so on.In KEGG pathway enrichment analysis , profilePRDM10, MYC, HIF1α, JUNB, and GAPDH), three , four , and four  hub genes were screened from profiles 4, 3, 0, and 7, respectively. The 16 hub genes had relatively high connectivity with other DEGs in the profiles, which suggests that these genes may play a relatively important role in response to acute high-temperature stress.The hub genes were screened from four significant profiles  by protein interaction network analysis. As shown in HSP90AB1), Ubiquitination-Proteasome , and HIF1A signaling pathway  for qPCR. As shown in To verify the DEGs identified by RNA-seq, we randomly selected 11 genes involved in HSPs , energy metabolism (glycolysis/gluconeogenesis and fatty acid biosynthesis), etc. play very important roles in high-temperature adaptation.Based on RNA-Seq technology, 1,347 DEGs and four significant profiles were screened from the transcriptomic data of 9 liver samples of rocesses , transcrAcute high-temperature stress can cause a cellular stress response in fish, leading to misfolding of intracellular proteins. If misfolded proteins accumulate in the body, it harms cells and interferes with their regular physiological functions . HSPs arHSP70 and HSP90AB1 in the T. ovatus liver significantly increased, and the increase was positively correlated with temperature. HSP70 is the most important member of the HSP family, with a molecular weight of around 70 KD. It is not expressed or expressed only in small amounts in cells under normal conditions, while its expression increases rapidly under stress conditions [HSP90AB1 gene and participates in the regulation of cell signal transduction, protein folding, and long-term cell adaptability [HSP70 and HSP90AB1 in the liver of T. ovatus are helpful for the refolding of misfolded proteins in cells, maintaining protein homeostasis and regular physiological functions, and minimizing damage to the fish body during heat stress. Consistent with this result, as the water temperature increased, the expression levels of HSP70 and HSP90 were also enhanced in brown trout  skin tissue [Ctenopharyngodon idellus) liver tissue [Lateolabrax maculatus) liver and muscle tissue [O. mykiss head kidney and brain tissue [Platichthys stellatus) kidney tissue [Coregonus clupeaformis) liver tissue [In this study, under acute heat stress, the expression of nditions . This prnditions . HSP90 itability . The uprn tissue , grass cr tissue , spottede tissue , O. mykin tissue ,64, stary tissue , and lakr tissue . This reIn addition to HSPs, another important means to regulate protein homeostasis and function in eukaryotic cells is UPS, and the degradation of more than 80% of proteins in cells depends on this system . UsuallyT. ovatus liver, namely, ubiquitin-conjugating enzyme E2D4 (UBE2D4), ubiquitin-protein ligase E3B (UBE3B), photomorphogenic regulatory factor 1 (COP1), and ring finger and CHY zinc finger domain containing 1 (RCHY1) after acute heat stress treatment. The UBE2D4 gene belongs to the ubiquitin-conjugating enzyme E2 gene family and encodes the ubiquitin-conjugating enzyme (E2), which acts as an intermediate to deliver activated ubiquitin molecules to E3 connected to the target protein [UBE3B, COP1, and RCHY1 genes encode ubiquitin ligase E3, a key enzyme in the ubiquitination reaction that binds the correct E2 and the substrate to increase the rate of ubiquitin transfer [Pachycara brachycephalum) was suppressed at high temperatures [2 and high-temperature stress, the gill tissues of blue-green damselfish (Chromis viridis) [O. mykiss [T. ovatus suggests that the activities of E2 and E3 may be reduced due to acute high-temperature stress, which slows down the ubiquitination process of target proteins (denatured or misfolded ones).The recognition and degradation of many proteins in cells depend on ubiquitination. This study found four significantly downregulated ubiquitination-related genes in  protein ,70. Thistransfer ,72. The eratures , consistviridis)  and O. m. mykiss  showed a. mykiss . E2 and PSMD3), non-ATPase 8 (PSMD8), non-ATPase 1 (PSMD1), ATPase 4 (PSMC4), and ATPase 5 (PSMC5) in T. ovatus liver were significantly downregulated at 34 °C. PSMD3 and PSMD8 encode two subunits in the lid; PSMD1, PSMC4, and PSMC5 encode three subunits in the base. Some studies have indicated that the decreased expression of proteasome subunits leads to the loss of proteasome activity [T. ovatus, downregulation of PSMD3, PSMD8, PSMD1, PSMC4, and PSMC5 may reduce the de-ubiquitination and protein unfolding abilities of 19S RP and its protein transfer function of presenting deubiquitinated and unfold proteins to 20SCP, slowing down the subsequent protein degradation process. 20S CP is the catalytically active part of the 26S proteasome and is responsible for recognizing, stretching, and degrading deubiquitinated proteins [PSMA4), alpha 7 (PSMA7), beta 3 (PSMB3), and beta 7 (PSMB7) in T. ovatus were significantly downregulated at 34 °C, which may cause the reduction or loss of 20S CP catalytic activity. That is, the degradation of denatured or misfolding proteins was likely to slow down. In C. idellus intestinal mucosa and O. mykiss gill tissue under the conditions of oxidized fish oil and high CO2 concentration, respectively, PSMA4, PSMB3, and PSMB7 were significantly upregulated, increasing the activity of 26S proteasome and helping to clear the damaged or incorrect proteins to protect tissues and cells from impairment [C. idellus infected with saprolegniasis, resulting in enhanced activity of 26S proteasome [C. idellus head kidney tissue responded to high-density and saline-alkali stress mainly in negative regulation [T. ovatus may lead to lower levels of proteasome subunits, leading to the reduction or loss of 26S proteasome activity.Different Ub molecules specifically mark the target protein, and the target protein marked by Ub will be degraded into small peptides or amino acids by the 26S proteasome . The 26Sactivity . Therefoproteins . It mainpairment ,82. The oteasome ,84. Genegulation , similarT. ovatus is inhibited to a certain extent. The degradation rate of misfolded proteins is reduced, allowing sufficient time for the refolding of misfolded proteins and the quality control of candidate degrading proteins. Under high-temperature stress, the concentration of DO in water decreases, and energy metabolism undergoes compensatory changes , oxygen transport , and energy metabolism  in the liver of T. ovatus showed significant changes. These changes may alter the body’s oxygen-carrying capacity and energy metabolism, resulting in adaptation to the hypoxic environment generated by acute heat stress.Water temperature is a crucial factor affecting aquatic organisms’ metabolic rate and water’s DO content. With the increase in water temperature, the metabolism of aquatic organisms is enhanced, and the oxygen consumption rate is also significantly increased. However, the temperature increase may reduce hemoglobin’s binding ability to oxygen . In addiHIF-1 is a key molecule regulating the hypoxic response of tissue cells and is composed of HIF-1α and HIF-1β subunits [HIF-1α is the active subunit of HIF-1. This subunit is widely present in most tissues, which are regulated by hypoxic signals and can regulate the activity of HIF-1 [HIF-1α depends on the oxygen concentration in the cell. Under normoxic conditions, HIF-1α is rapidly degraded through UPS, but under hypoxic conditions, HIF-1α will continue to accumulate and bind to HIF-1β inside the nucleus, forming active HIF-1 [HIF-1α in the liver of T. ovatus decreased at 31 °C but was significantly upregulated at 34 °C. Most tissues of channel catfish  [Danio rerio) [HIF-1α (decreased first and then increased) during hypoxic stress. During the early stage of hypoxia, the expression of HIF-1α was not upregulated but downregulated, which may be because HIF-1 gradually accumulated during the early stage, and this accumulation then negatively regulated gene transcription [HIF-1α [HIF-1α was significantly upregulated in D. rerio embryos [Astronotus ocellatus) livers [Pelteobagrus fulvidraco) livers [O. mykiss kidney [HIF-1α in T. ovatus. HIF-1α further forms different signaling pathways with various upstream and downstream proteins, mediating hypoxic signals and regulating a series of compensatory responses to hypoxia in cells.subunits . Of thesof HIF-1 . The effve HIF-1 . This stnctatus)  and the o rerio)  showed acription . Other r [HIF-1α ,95. Unde embryos , oscar  livers , yellow ) livers , O. mykis kidney , etc. ItHIF-1α is an important factor in regulating EPO expression [EPO, a downstream gene of HIF-1α, was slightly downregulated at 31 °C but significantly upregulated at 34 °C, which was consistent with the expression trend of HIF-1α. EPO is a glycoprotein hormone that can stimulate the differentiation and proliferation of erythroid precursor cells [EPO may improve the differentiation of red blood cells in the liver, effectively increase the number of red blood cells and hemoglobin, and then increase the body’s O2-carrying capacity and utilization of oxygen to compensate for the body’s hypoxia state in a high-temperature hypoxic environment [EPO’s expression level helps T. ovatus effectively use low oxygen concentrations and maintain normal physiological processes.pression . This stor cells . It is aor cells . The uprironment ,103. TheEPO gene, the upregulated HIF-1α can also regulate the transcription of downstream glycolysis-related genes [PFKL, ALDOB, GAPDH, and ENO1) in the liver of T. ovatus showed significant upregulation trends. Among them, PFKL (a glycolytic rate-limiting enzyme) plays a decisive role in regulating the direction and rate of the glycolytic metabolic pathway and can catalyze the reaction of fructose-6-phosphate (F6P) to generate fructose-1,6-phosphate . Both ALDOB and GAPDH are key enzymes of glycolysis, and ENO1 is the rate-limiting enzyme of glycolysis. F1,6P is gradually converted into phosphoenolpyruvate (PEP) with enzymes, such as ALDOB, GAPDH, and ENO1, which promotes the generation of more pyruvate to participate in the tricarboxylic acid cycle or other metabolic pathways. In this study, the significant upregulation of PFKL, ALDOB, GAPDH, and ENO1 expressions indicates that in a high-temperature and low-oxygen environment, anaerobic glycolysis is activated in the liver of T. ovatus, becoming one of the primary energy supply pathways for the body. Similar findings have been reported in previous fish studies. In O. Niloticus [Micropterus salmoides) [Larimichthys crocea) [In addition to the ed genes ,105, whiiloticus , largemolmoides) , and lar crocea) , the livACSL1), long-chain acyl-CoA synthetase 3 (ACSL3), fatty acid desaturase 2 (FADS2), and elongase of very long chain fatty acid 6 (ELOVL6) were significantly downregulated in the liver of T. ovatus at 34 °C, implying that fatty acid absorption and unsaturated fatty acids synthesis may be reduced. The long-chain fatty acyl-CoA synthetases (ACSLs) family is a crucial enzyme in the synthesis and catabolism of fatty acids and can catalyze the formation of acyl-CoA from free fatty acids (an energy-consuming process) [ACSL1 and ACSL3 are important members of the ACSLs family, and they can accelerate the synthesis and catabolism of fatty acids. ACSL1 can transport saturated fatty acids and unsaturated fatty acids into cells and catalyze them, and this protein is highly expressed in tissues related to energy metabolism, such as fat and liver tissues [C. idellus, ACSL1 catalyzes the acylation of endogenous fatty acids, activating the β-oxidation pathway and providing energy for fish [ACSL3 is a lipid droplet-associated protein that participates in the absorption of fatty acids and helps maintain lipid homeostasis. Under an acute hyperthermic hypoxic environment, the significant downregulation of ACSL1 and ACSL3 in the liver of T. ovatus may hinder fatty acid absorption and β-oxidation, thereby reducing the liver tissue’s demand for O2. With the enhanced glycolysis, transcriptome sequencing results showed that the fatty acid metabolism-related genes long-chain acyl-CoA synthetases 1 (process) . ACSL1 a tissues ,111. In for fish . ACSL3 iFADS2 and ELOVL6, belonging to the FADS and ELOVLs families, respectively, are rate-limiting enzymes in synthesizing unsaturated fatty acids and catalyzing fatty acid dehydrogenation and carbon chain elongation reactions respectively. In this study, the significant downregulation of expression levels of FADS2 and ELOVL6 indicates that fatty acid dehydrogenation and carbon chain elongation were inhibited in the liver of T. ovatus. That is, the body’s ability to synthesize unsaturated fatty acids was reduced. Under high-temperature stress, the expression levels of genes related to unsaturated fatty acid syntheses, such as FADS2, ELOVL2, and ELOVL5, were also significantly decreased in the muscle of fathead minnows  [2 and energy to adapt to the hypoxic environment and compensatory changes in energy metabolism caused by high temperature.In addition, the formation of unsaturated fatty acids is an oxygen-consuming process, which uses fatty acid chains as substrates to synthesize unsaturated fatty acids through a series of alternating reactions of dehydrogenation and carbon chain elongation under the catalysis of FADS and ELOVLs ,114. FADromelas) . It can HIF-1 signaling pathway in T. ovatus, which in turn regulates the cells to produce a series of compensatory responses to hypoxia, such as enhancing O2-carrying ability and O2-utilization rate, activating the anaerobic glycolysis pathway, and inhibiting fatty acid metabolism. These responses are conducive to the body’s effectively using low-concentration oxygen to maintain normal energy metabolism and other physiological processes to adapt to hypoxia caused by a high-temperature environment.In summary, as the water temperature increases, the DO in the water decreases, and the continuous hypoxic environment activates the T. ovatus. Under acute heat stress, the expression of HSPs was increased, and the expression of UPS was inhibited, which helped to reduce the aggregation of denatured and misfolded proteins and further maintain normal protein and liver physiological functions. Moreover, the HIF-1 signaling pathway was activated by a hypoxic environment caused by acute heat stress, further inducing a series of compensatory responses, such as the increase in oxygen-carrying ability and oxygen utilization, activation of the anaerobic glycolytic pathway, inhibition of fatty acid metabolism, and so on, which helps effective utilization of low concentration of oxygen at the organismal level to maintain normal energy metabolism and other physiological processes. The above findings indicated that maintaining normal protein function, energy metabolism, and other physiological functions contribute to heat stress adaption. The present study preliminary explores the molecular response mechanism of T. ovatus under acute heat stress. These genes, pathways, and biological processes related to high temperature screened in this study would be of great value for selecting and breeding heat-tolerant cultivars and the high-quality development of T. ovatus farming.Through liver transcriptomic analysis, our studies found that genes and biological processes related to HSPs, UPS, HIF-1 signaling pathway, and energy metabolism (glycolysis and fatty acid metabolism) play a critical role in response to heat stress in the liver of


Cancer survivors undergone treatment face an increased risk of developing atherosclerotic cardiovascular disease (CVD), yet the underlying mechanisms remain elusive. Recent studies have revealed that chemotherapy can drive senescent cancer cells to acquire a proliferative phenotype known as senescence-associated stemness (SAS). These SAS cells exhibit enhanced growth and resistance to cancer treatment, thereby contributing to disease progression. Endothelial cell (EC) senescence has been implicated in atherosclerosis and cancer, including among cancer survivors. Treatment modalities for cancer can induce EC senescence, leading to the development of SAS phenotype and subsequent atherosclerosis in cancer survivors. Consequently, targeting senescent ECs displaying the SAS phenotype hold promise as a therapeutic approach for managing atherosclerotic CVD in this population. This review aims to provide a mechanistic understanding of SAS induction in ECs and its contribution to atherosclerosis among cancer survivors. We delve into the mechanisms underlying EC senescence in response to disturbed flow and ionizing radiation, which play pivotal role in atherosclerosis and cancer. Key pathways, including p90RSK/TERF2IP, TGFβR1/SMAD, and BH4 signaling are explored as potential targets for cancer treatment. By comprehending the similarities and distinctions between different types of senescence and the associated pathways, we can pave the way for targeted interventions aim at enhancing the cardiovascular health of this vulnerable population. The insights gained from this review may facilitate the development of novel therapeutic strategies for managing atherosclerotic CVD in cancer survivors. AtherosCancer survivors who have undergone cancer treatment, including radiation therapy, face an elevated risk of developing atherosclerotic CVD, with a 1.3–3.6 fold increase in developing coronary artery disease (CAD) and a 1.7–18.5-fold increase in developing atherosclerotic risk factors, which may lead to potentially fatal consequences . This inCellular senescence, a hallmark of aging, occurs when cells cease to divide in response to various stress stimuli, both internal and external. These stimuli include d-flow, oncogene activation, DNA damage, mitochondrial dysfunction, reactive oxygen species (ROS), and cancer treatments including radiation therapy and chemotherapy. Recent study by Milanovic and colleagues has shown that chemotherapy may induce senescence in cancer cells, which can have negative effects on tissue functions due to the secretion of various factors that regulate vital biological processes, such as cellular metabolism, cell growth, and inflammatory signaling , 10. ThiMilanovic and colleagues' seminal observation revealed clear differences between replicative senescence (RS) and stress-induced premature senescence (SIPS) –16. SIPSCancer treatment can also lead to changes in the structure and function of the vasculature, including alterations in the morphology and function of ECs that can induce EC senescence . EC seneIn this review, we aim to provide a comprehensive understanding of the possible molecular mechanisms regulating the development of atherosclerotic CVD in cancer survivors to establish rational therapeutic directions based on a mechanistic understanding of their interactions and interplay between cancer therapy and hemodynamics. We will summarize the similarities and differences between RS and SIPS, the latter of which plays a critical role in the induction of the SAS phenotype. We will describe the importance of the regulatory mechanisms for telomere DNA protection, as telomere DNA damage and dysfunction are critical for the induction of SIPS. We will also examine the key pathways involved in cellular senescence. Finally, we will discuss potential therapeutic interventions that may benefit patients with atherosclerotic CVD, with a focus on the BH4 pathway.Our goal is to provide current knowledge to help future studies understand how cellular senescence is triggered in ECs in response to cancer treatments and how a distinct type of SASP (SAS) in ECs may contribute to the development of atherosclerotic CVD in cancer survivors.Additionally, we aim to highlight the potential of senolytic drugs in preventing, delaying, and alleviating various age-related disorders, including atherosclerotic CVD, in humans .2.2.1.Atherosclerosis is a chronic and progressive inflammatory disease that involves various risk factors, including d-flow and elevated level of oxidized low-density lipoprotein (oxLDL) . It can Recent studies have also suggested that cellular senescence may play a role in plaque destabilization and rupture, leading to acute cardiovascular events. For instance, senescent vascular smooth muscle cells (VSMCs) within the plaque have been shown to have increased matrix metalloproteinase (MMP) activity, which can contribute to plaque rupture and thrombosis. Additionally, senescent cells can secrete SASP, characterized by the release of numerous molecules including proinflammatory cytokines and chemokines, such as interleukine 1β (IL1β), tumor necrosis factor α (TNFα), interferon γ (IFNγ), and transforming growth factor β (TGFβ). The SASP can lead to chronic inflammation by inducing the persistent pro-inflammatory senescence phenotype (PISP), promoting the recruitment and activation of immune cells within the plaque, impairing cholesterol efflux from macrophages, and leading to the accumulation of lipid-laden foam cells in the plaque –32.2.2.Cellular senescence can have a dual role in cancer, both suppressing and promoting its development and progression . On one Over time, the accumulate of senescent cells in tissues can lead to chronic inflammation, tissue dysfunction, and organ failure, all of which can increase the risk of cancer development . Several2.3.Milanovic and colleagues have demonstrated that chemotherapy can drive a specific subset of senescent cancer cells to reprogram and acquire a proliferative phenotype known as SAS. This unique state allows these cells to bypass cell cycle arrest and significantly enhances their growth and proliferation –16, indeThese findings highlight the significant role of cellular senescence in the development and progression of atherosclerotic CVD and cancer, including among cancer survivors. However, it is important to note that senescence is also associated with impaired angiogenesis . Studies3.3.1.Cellular senescence is a state of irreversible cell cycle arrest, first described by Hayflick and Moorhead in 1961 when they observed that human diploid fibroblasts underwent this process after serial passaging. Successive cell division can shorten telomeres to a critical length, known as the Hayflick limit –43, at wTelomeres are repetitive hexanucleotide 5′-TTAGGG-3′tandem sequences that cap the chromosome ends, responsible for maintaining genomic stability –43. On eTo prevent continuous shortening of telomeres, the telomerase protein, which is a telomere-specific ribonucleoprotein, adds single-stranded telomeric repeats to the chromosomal 3′ ends . HoweverCip1 (or p21Waf1) and p16INK4a  and cyclin-dependent kinase inhibitors p21p16INK4a , 57. Thip16INK4a .3.2.Telomere shortening has been linked to cellular senescence , but theAdditionally, studies have shown that cellular senescence is also a characteristic of chronic stress, which can provoke premature senescence, or so-called SIPS through internal and external stimuli –69. SIPSRS and SIPS are induced at different time frames and regulated by different mechanisms as reviewed elsewhere –47, 71. 3.3.DNA damage, as detected by the formation of g-H2AX foci (g-foci), has been linked to cellular senescence and aging at the organismal level. Both telomeric and non-telomeric DNA damage have been implicated in cellular senescence. Nakamura and colleagues have shown that g-foci related to senescence can be found at uncapped telomeres or non-telomeric DNA damage sites on chromosomes in both humans and mice . Telomer3.4.Senescent cells, including both RS and SIPS, are known to secrete a variety of molecules that contribute to pro-inflammatory and pro-tumorigenic environment, collectively referred to as SASP , 80–84. In addition to their effects of neighboring cells, SASP cells can also communicate via extracellular vesicles (EVs) . Activat3.5.Milanovic and colleagues reported that cancer therapy-induced senescence (TIS) can trigger the SAS proliferative phenotype in cancer cells, allowing them to evade senescence-induced cell cycle arrest and exhibit enhanced clonogenic growth potential , 16, 20.In addition to TIS, oncogene-induced senescence (OIS) is another phenomenon in which oncogenes are activated in non-tumor cells, resulting in a stable cell cycle arrest . Leon anIt is important to note that, although the findings observed by Milanovic and colleagues were made on cancer cells, it is possible that a similar phenomenon might also occur in vascular cells, including ECs. Both senescence and macrophage proliferation contribute to CVD , and the3.6.As we discussed above, cellular senescence has been implicated in the development of both atherosclerosis and cancer including in cancer survivors. At the molecular level, cellular senescence is associated with both telomeric and non-telomeric DNA damage in cells of the vessel wall, including ECs, triggering the DDR pathway –100. DDRThe major DDR pathways are regulated by the ataxia-telangiectasia mutated and ataxia-telangiectasia and Rad3 related (ATM/ATR) pathways, which phosphorylate proteins at DNA damage sites, including histone H2A (H2AX), to form phosphorylated gH2AX. Additionally, ATM/ATR activate CHK2 and CHK1, respectively , which p4.Cellular senescence can occur independently of telomere shortening, highlighting the importance of understanding the mechanisms that protect telomeres from DNA damage and dysfunction to mitigate senescence-associated disorders and diseases. In this section, we will explore the mechanisms involved in the regulation of telomere protection pathways.Chromosome ends must be shielded from damage to prevent the acceleration of cellular senescence, which would lead to activation of DDR and DNA damage repair machinery. This protection is accomplished by specific factors associated with telomeres, including telomerase, non-coding telomeric repeat-containing RNAs (TERRA), and Shelterin , 106.4.1.TERC and a reverse transcriptase enzymatic subunit TERT, catalyzes the addition of repetitive hexanucleotide 5′-TTAGGG-3′sequences to chromosome ends. This process known as telomere elongation  are two key components of vertebrate telomeres that share the TRF homology domain (TRFH) and bind double-stranded telomeric DNA as homodimers. TRFH on TRF1 and TRF2 is critical for TRF1-TRF2 dimerization, DNA binding, telomere localization, and modulation of TRF1-TRF2 interaction with other Shelterin proteins . To studBesides their central role in telomere capping, elevated TRF2 expression is frequently found in tumors. El Mai and colleagues have demonstrated that TRF2 is expressed in the vasculature of most human cancers, where it colocalizes with the Wilms' tumor suppressor (WT1). TRF2 acts as a transcriptional target of WT1 and plays an essential role in EC proliferation, migration, and tube formation. Mechanistically, TRF2 binds and transactivates the promoter of angiogenic tyrosine kinase platelet-derived growth factor receptor β (PDGFRβ) through a mechanism distinct from that of telomere capping .TRF1-interacting protein 2 (TIN2) is a key protein in the Shelterin complex, acting as a central mediator for TRF1 function. Using TRF1 as bait for interaction cloning, Kim and colleagues demonstrated that TIN2 interacts with TRF1 and co-localizes with it in the nucleus during metaphase chromosomes. TIN2 also binds telomere protection protein 1 (TPP1)/protection telomeres 1 (POT1) to regulate telomere length, telomeric capping, and telomerase activity , 126. POTTP1/POT1 are single-stranded telomeric DNA binding proteins that play essential roles in preventing the activation of the ATM/ATR pathways at telomeres –131. AltOf the six Shelterin proteins, TRF1-TRF2 bind double-stranded telomeric DNA through the C-terminal Myb domain and recruit TERF2IP, TIN2, TPP1, and POT1 for assembling the Shelterin complex . Among tThe Shelterin complex is a promising target for chemotherapeutics. 4.2.1.To bind double-stranded telomeric DNA, TRF1 and TRF2 associate with TIN2, which stabilizes the telomeric localization of TRF1 and TRF2. Although TRF1 and TRF2 share similarities in structure and form TRF1-TRF2 dimers, their key functions are distinct from each other , 142. TRTRF2, on the other hand, functions as a telomere capping protein and is involved in protecting chromosome ends from being recognized as DNA damage site, which can lead to genomic instability. In addition to its role in telomere attachment to nuclear membrane , TRF2 al4.2.2.TIN2 serves as an adaptor protein for TRF1 and helps in stabilizing the telomeric localization of TRF1 and TRF2 . Additio4.2.3.+)-dependent deacetylase, have been shown by Chen and colleagues to protect mesenchymal stem cells from senescence by mediating TPP1 expression, suggesting TPP1's involvement in cellular senescence . I. I165). 4.2.5.TERF2IP, also known as RAP1, plays a crucial role in several biological processes, including telomere protection, homology-directed repair (HDR) regulation, inflammation, and metabolism , 171. TE4.3.4.3.1.NO has a crucial role in various physiological and pathological processes in the human body, including protecting telomeres from shortening, damage, and dysfunction by regulating telomerase activity, thus protecting cells from senescence. Additionally, NO can prevent DNA damage and oxidative stress, which can also contribute to telomere shortening and dysfunction. Exogenous NO has been demonstrated to delay EC senescence in culture, suggesting that telomere shortening can be modulated beyond the number of cellular divisions. The mechanism by which NO stimulates telomerase activity is not yet fully understood, but it may react with tissue-derived oxygen radicals, thereby reducing oxidative stress, which has been shown to accelerate EC senescence. Alternatively, NO may upregulate telomerase activity via transcriptional and/or posttranscriptional mechanisms .NO has important cardioprotective and anti-inflammatory effects, as it inhibits vascular wall apoptosis and lipid oxidation, prevents VSMC growth, and inhibits white blood cell adhesion and platelet aggregation. Additionally, NO induces blood vessel dilation, which is a vital mediator of vascular tone , 182. ThConversely, endothelial NO is essential for angiogenesis and protects against atherosclerosis. However, the bioavailability of endothelial-derived NO is impaired with aging, which may accelerate EC senescence, impairing EC function and contributing to impaired angiogenesis and atherosclerotic progression , 183–1864.3.2.BH4 is an essential cofactor involved in several important enzymatic processes. It plays a vital role in the synthesis of neurotransmitters such as adrenaline, noradrenaline, serotonin, and dopamine, and participate in the degradation of phenylalanine . Additiode novo synthesis from guanosine triphosphate (GTP) and a salvage pathway that involves recycling BH2 and quinoid-dihydrobiopterin , which can stimulate the production of higher levels of superoxide by eNOS. This oxidative process can lead to increased oxidative stress and potentially impact EC function . ECs haviopterin , 191. ThAge-related studies have shown a decrease in vascular BH4 levels in animal models , 193, wh5.It is noteworthy that senescent ECs have been identified in human atherosclerotic plaques and have impaired function, such as reduced NO production and increased adhesion molecule expression, which can contribute to EC dysfunction and plaque formation , 174. ThThe impairment of EC functions is an initial step in the progression of atherosclerotic CVD, which is linked to the aging process and a senescent phenotype in these cells. This can lead to disrupted permeability and pathological signaling cues, as evidenced by numerous studies , 203–205Moreover, it is crucial to note that radiation therapy, a commonly cancer treatment, directly induces endoMT , which i5.1.ECs can undergo a process called endoMT, which leads to their trans-differentiation into mesenchymal cells , 212. DuInflammatory mediators such as IL1β, TNFα, and NFκB cause EC dysfunction, leading to endoMT, which is the link between inflammation and EC inflammation-associated disorders . Recent 5.1.1.In vitro studies have revealed that various processes contributing to atherosclerosis, including TGFβ signaling, oxidative stress, and hypoxia, can activate endoMT  and five ligands (delta-like ligand 1/3/4 (DLL1/3/4) and Jagged 1/2 (JAG1/2)) . Upon li5.3.SUMOylation is involved in regulating many essential cellular processes, including those in the nucleus such as transcriptional activity, chromatin remodeling, and DDR , 260–2635.3.1.TGFβ signaling is regulated at various levels, including TGFβR activation and post-translation modifications . TGFβ SUThe TGFβ signaling cascade begins with TGFβR1/2 dimerization followed by TGFβ activation. Activated TGFβR1/2 promotes TGFβR1 K389 SUMOylation, which enhances TGFβR-SMAD association and SMAD2/3 phosphorylation . TGFβR S5.3.2.The SMAD family of proteins is divided into three subfamilies: receptor-activated SMADs , common mediator SMADs (Co-SMADs), and inhibitory SMADs (iSMADs including SMAD6/7) . R-SMADs6.ECs play a crucial role as transducers between blood flow mechanics and cellular signaling pathways in both physiological and pathological conditions. Mechano-sensors on the surface of ECs sense changes in blood flow and transduce these mechanical forces into biochemical signals, subsequently activating cellular responses that regulate downstream events . Hemodyn6.1.6.1.1.In regions of l-flow, ECs aligned longitudinally in the direction of blood flow and secrete factors for vasodilation and anticoagulation. L-flow promotes the generation of NO and expression of downstream molecules that improve vascular injury and suppress inflammation, making it an atheroprotective mechanical force that maintain EC homeostasis and function . This isStudies on cultured human aortic ECs have shown that l-flow downregulates proliferation- and inflammation-related genes and upregulates survival-, angiogenesis-  and vascular remodeling-  related genes . This suL-flow increases NO levels immediately through stimulation of eNOS, and long-term exposure can lead to increased mRNA and protein expression of eNOS. Acute eNOS activation could result from a shear-stress-derived intracellular calcium increase, which enhances calmodulin binding to eNOS. Nonetheless, eNOS phosphorylation by various phosphorylases, including PI3K/AKT, adenylate cyclase, and protein kinase A (PKA) have also been involved in the rapid response to l-flow . Long-teFurthermore, the resulting high shear stress has been found to increase BH4 levels by stimulating the first and rate-limiting step of its synthesis, GCH1. L-flow resulted in a ∼30-fold increase in GCH1 activity produced through phosphorylation of serine 81 by the α' subunit of casein kinase 2 (CK2), but did not produce increased protein expression, indicating BH4's short-term role in l-flow-associated NO synthesis. This, in turn, increases levels of NO and promotes an overall cardioprotective effect .6.1.2.In regions of d-flow, ECs undergo several changes including becoming polygonal and poorly aligned and undergo senescence and apoptosis. Moreover, they also secrete factors that lead to vasoconstriction and coagulation, promoting the generation of ROS, platelet aggregation, EC activation, senescence, inflammation, dysfunction, permeability, and endoMT as depicted in 6.1.3.D-flow and l-flow have different effects on the accessibility of transcription factor binding motifs and cis-regulatory elements, leading to differential regulation of EC-specific gene expression at both genomic and epigenomic levels. Downstream factors, such as kruppel-like factors 2/4 (KLF2/4), are enriched in ECs exposed to l-flow, while known transcription factor binding motifs  and novel transcription factor binding motifs  are enriched in ECs exposed to d-flow .Research using single-cell RNA-seq and single-cell assay for transposase accessible chromatin sequencing (ATACseq) in mice after partial carotid ligation showed that d-flow can reprogram ECs from athero-protective cells to atherogenic proinflammatory cells, endoMT cells, hematopoietic stem cells, endothelial progenitor cells, and cells with immune cell-like phenotypes . Mechaniin vivo and in vitro studies, Mahmoud and colleagues have demonstrated that d-flow upregulates the expression of twist family bHLH transcriptional factor 1 (TWIST1) via a mechanism regulated by the developmental transcription factor GATA4. Microarray data revealed that both GATA4 and TWIST1 expression were enriched in ECs exposed to d-flow regions in the porcine aorta in vivo. In vitro studies using controlled flow systems also showed that GATA4 and TWIST1 expression were enhanced in cultured ECs exposed to d-flow compared to non-d-flow conditions , can decrease the expression of fibroblast growth factor receptor 1 (FGFR1) in ECs, leading to activation of downstream TGFβ signaling and subsequent endoMT activation . StudiesZhao and colleagues used a three-dimensional micro-engineered human coronary artery-on-a-chip model to demonstrate that d-flow drives ECs towards a proinflammatory endoMT phenotype, accompanied by elevated expression of VCAM1 and ICAM1, which can mediate recruitment of monocytes, leukocytes, and macrophages to plaque sites , 311. Ac6.1.4.2–), a potent ROS, and pro-atherosclerotic vascular dysfunction  through the bloodstream. The model includes stochastic adhesion events and uses a realistic model of global blood circulation to simulate cancer cell trajectories. The authors compared the model's predictions with data from thousands of human autopsies for seven different solid tumors, including lung, prostate, pancreatic, and colorectal cancers. They found that, on average, 40% of the variation in the metastatic distribution could be attributed to blood circulation –328.6.3.Activation of the transcription factor NFκB is critical for initiating an inflammatory genetic program critical that contributes to atherogenesis, which is triggered by d-flow , 330. GaD-flow induces various posttranslational modifications that promote inflammatory signaling, including SUMOylation . SUMOylaKnockout of SENP2 in mice leads to developmental defects in trophoblast stem cell niches and lineages due to dysregulation of the Mdm2-p53 signaling pathway in the placenta . In huma7.7.1.Atherogenesis predominantly occurs in regions of vascular ECs exposed to d-flow but not l-flow , 345–349Similarly, many cancer treatments, including radiation therapy and chemotherapy, can induce cellular senescence, a phenomenon known as TIS , 354. Wh7.2.de novo biosynthesis  and protein kinase B , while d-flow activates only PKB , 363. InRadiation therapy is a common treatment modality for solid tumors , with ovTo investigate the molecular mechanism underlying atherosclerotic CVD development after thoracic radiotherapy, Kim and colleagues exposed human aortic ECs to 5Gy of IR for 24 h and observed an increased expression of fibroblast markers αSMA and FSP1 accompanied by a decreased expression of EC markers CD31 and VE-cad. The authors also noted that oxLDL increases IR-induced endoMT, which contributes to atherosclerotic plaque formation . SimilarRecent research by Choi and colleagues reported that in human umbilical vein ECs exposed to radiation, TRP53 expression increases while TGFβ2 decreases endoMT. siRNA-mediated TRP53 silencing abrogated radiation-induced endoMT, resulting in a decreased expression of endoMT-related transcription factors such as SNAIL1/2 and zinc finger E-box-binding homeobox 2 (ZEB2) . These f7.4.We have found that d-flow activates p90RSK , 335, whFurthermore, we have discovered that cytoplasmic SENP2 plays a role in inhibiting the SUMOylation of the membrane-associated protein MAGI1, which is a tight and adherent junction protein with a newly identified role in EC function and atherosclerosis . Recentl7.5.Post-translational modifications are crucial for regulating the cellular localization and function of TERF2IP, which is necessary for protecting telomeres , 377. OuNotably, TERF2IP expression is increased in d-flow regions of atherosclerotic plaques and has been linked to various cancers, such as breast cancers, gastric carcinoma, non-small cell lung cancer, and mantle cell lymphoma , 382–384In a study using mice that received 2 Gy whole-body γ-radiation, it was found that VCAM1 expression is upregulated in the d-flow, further implicating the role of p90RSK-mediated TERF2IP S205 phosphorylation in the progression of atherosclerotic CVD progression induced by d-flow and chemoradiation therapies , 377.8.The accumulation of senescent cells during RS or SIPS is one of the challenges in the treatment of atherosclerotic CVD and other age-related diseases. Therefore, eliminating senescent cells using senolytic drugs or attenuating the SASP without inducing apoptosis of senescent cells using senomorphic drugs is considered a potential treatment strategy nowadays. 9.Our aim with this review is to offer a comprehensive overview of the interactions between multiple key components that contribute to SIPS and the development of atherosclerotic CVD in cancer survivors. We intend to identify potential therapeutic targets for the prevention and treatment of atherosclerotic CVD and provide new insights into the molecular mechanisms underlying atherosclerosis development in cancer survivors. We believe that the information presented in this review will be beneficial to a diverse audience of researchers and clinicians in the fields of cardiology, oncology, and molecular biology, as well as to patients and their families who may be concerned about the long-term health effects of cancer treatment. Finally, we emphasize the need for further research, particularly in ECs, to gain a better understanding of the mechanisms involved in the development of atherosclerosis in cancer survivors after receiving cancer therapy.A potential link between BH4 and eNOS uncoupling has been proposed, but the precise mechanism underlying this process remains unclear . PreventThe impact of altered hemodynamic shear stress, either alone and in combination with cancer treatment, on DDR signaling pathways and SIPS in cancer survivors is not well understood and requires further investigation. However, it is possible that d-flow and cancer therapy accelerate premature senescence through p90RSK-driven posttranslational modifications of TERF2IP and disruption of the Shelterin complex, which is formed by TRF1, TRF2, TIN2, TPP1, POT1, and TERF2IP/RAP1 , 122. Al