Design and optimization of piperlongumine analogs as potent senolytics.

Selective removal of senescent cells (SnCs) offers a promising therapeutic strategy to treat chronic and age-related diseases. Our prior investigations led to the discovery of piperlongumine (PL) and its derivatives as senolytic agents. In this study, our medicinal chemistry campaign on both the α,ß-unsaturated δ-valerolactam ring and the phenyl ring of PL culminated in the identification of compound 24, which exhibited an impressive 50-fold enhancement in senolytic activity against senescent WI-38 fibroblasts compared to PL.

Effect of HIF1α on the TRPC6 channel of glomerular podocytes under chronic hypoxia.

BACKGROUND: Chronic hypoxia plays an important role in the initiation and progression of chronic renal disease. The pathogenic role of chronic hypoxia in tubulointerstitial injury has been investigated widely, but little is known about acute hypoxia implications in glomerular damage. In this study, we investigated the effect of chronic hypoxia on transient receptor potential cation channel 6 (TRPC6) and the underlying mechanism in cultured human podocytes. METHODS: Fluo-3 was used as a calcium indicator of the OAG-induced receptor operated calcium entry (ROCE) and basal [Ca2+]i levels were monitored using laser scanning confocal microscope after exposure of cells to chronic hypoxia. 2-aminoethoxydiphenylborane (2-APB), a pharmacological blocker of TRPCs channels, was used to determine the role of TRPC6 in podocytes under chronic hypoxia. The mRNA expression and protein levels of TRPC6 were determined using Real-time RT-PCR and Western Blotting under normoxic and chronic hypoxic conditions. Actin arrangement was analyzed by confocal microscopy using phalloidin staining of F-actin in podocytes. RESULTS: Cytosolic free Ca2+ was increased by hypoxia or the treatment of TRPC6 agonist OAG under normoxic conditions. The increase of intracellular Ca2+ induced by hypoxia was time- and dose-dependent, which can be inhibited by 2-APB, demonstrating that the changes of intracellular Ca2+ induced by OAG depend on the activation of TRPC6. Further study showed that the TRPC6 expression levels were significantly increased by hypoxia, which were inhibited by the HIF1α inhibitor in podocytes. Similarly, the increase of intracellular Ca2+ induced by hypoxia was decreased when the podocytes were incubated with HIF1α inhibitor. We also found that F-actin was ruptured by hypoxia in podocytes, showing cytoskeleton reorganization. CONCLUSIONS: TRPC6 mRNA and protein expression levels were significantly increased in podocytes under hypoxia, which may result in the increase of intracellular Ca2+. This alternation of TRPC6 may be relevant to the modulation of HIF1α. Hypoxia in podocytes can result in cytoskeleton reorganization, which further leads to podocytes injury and disfunction.

Transcriptome evidence reveals enhanced autophagy-lysosomal function in centenarians.

Centenarians (CENs) are excellent subjects to study the mechanisms of human longevity and healthy aging. Here, we analyzed the transcriptomes of 76 centenarians, 54 centenarian-children, and 41 spouses of centenarian-children by RNA sequencing and found that, among the significantly differentially expressed genes (SDEGs) exhibited by CENs, the autophagy-lysosomal pathway is significantly up-regulated. Overexpression of several genes from this pathway, CTSB, ATP6V0C, ATG4D, and WIPI1, could promote autophagy and delay senescence in cultured IMR-90 cells, while overexpression of the Drosophila homolog of WIPI1, Atg18a, extended the life span in transgenic flies. Interestingly, the enhanced autophagy-lysosomal activity could be partially passed on to their offspring, as manifested by their higher levels of both autophagy-encoding genes and serum beclin 1 (BECN1). In light of the normal age-related decline of autophagy-lysosomal functions, these findings provide a compelling explanation for achieving longevity in, at least, female CENs, given the gender bias in our collected samples, and suggest that the enhanced waste-cleaning activity via autophagy may serve as a conserved mechanism to prolong the life span from Drosophila to humans.

Maternal high folic acid supplement promotes glucose intolerance and insulin resistance in male mouse offspring fed a high-fat diet.

Maternal nutrition may influence metabolic profiles in offspring. We aimed to investigate the effect of maternal folic acid supplement on glucose metabolism in mouse offspring fed a high-fat diet (HFD). Sixty C57BL/6 female mice were randomly assigned into three dietary groups and fed the AIN-93G diet containing 2 (control), 5 (recommended folic acid supplement, RFolS) or 40 (high folic acid supplement, HFolS) mg folic acid/kg of diet. All male offspring were fed HFD for eight weeks. Physiological, biochemical and genetic variables were measured. Before HFD feeding, developmental variables and metabolic profiles were comparable among each offspring group. However, after eight weeks of HFD feeding, the offspring of HFolS dams (Off-HFolS) were more vulnerable to suffer from obesity (p=0.009), glucose intolerance (p<0.001) and insulin resistance (p<0.001), compared with the controls. Off-HFolS had reduced serum adiponectin concentration, accompanied with decreased adiponectin mRNA level but increased global DNA methylation level in white adipose tissue. In conclusion, our results suggest maternal HFolS exacerbates the detrimental effect of HFD on glucose intolerance and insulin resistance in male offspring, implying that HFolS during pregnancy should be adopted cautiously in the general population of pregnant women to avoid potential deleterious effect on the metabolic diseases in their offspring.

Revolutionizing Diabetic Foot Ulcer Care: The Senotherapeutic Approach.

Diabetic foot ulcers (DFUs) are a prevalent and profoundly debilitating complication that afflicts individuals with diabetes mellitus (DM). These ulcers are associated with substantial morbidity, recurrence rates, disability, and mortality, imposing substantial economic, psychological, and medical burdens. Timely detection and intervention can mitigate the morbidity and disparities linked to DFU. Nevertheless, current therapeutic approaches for DFU continue to grapple with multifaceted limitations. A growing body of evidence emphasizes the crucial role of cellular senescence in the pathogenesis of chronic wounds. Interventions that try to delay cellular senescence, eliminate senescent cells (SnCs), or suppress the senescence-associated secretory phenotype (SASP) have shown promise for helping chronic wounds to heal. In this context, targeting cellular senescence emerges as a novel therapeutic strategy for DFU. In this comprehensive review, we look at the pathology and treatment of DFU in a systematic way. We also explain the growing importance of investigating SnCs in DFU and highlight the great potential of senotherapeutics that target SnCs in DFU treatment. The development of efficacious and safe senotherapeutics represents a pioneering therapeutic approach aimed at enhancing the quality of life for individuals affected by DFU.

Transcriptomic and intervention evidence reveals domestic dogs as a promising model for anti-inflammatory investigation.

Domestic dogs have great potential to expand our understanding of the determinants of aging. To understand the aging pattern of domestic dogs and evaluate whether they can be used as an aging model, we performed RNA sequencing on white blood cells from domestic dogs aged 1-9 years and treated aged dogs with classical antiaging approaches. We obtained 30 RNA sequencing libraries and identified 61 age-associated genes with dynamic changes, the majority of which were related to metabolism and immune function, which may be predominant biomarkers for aging in dogs. We next treated aged dogs with canine mesenchymal stem cells (cMSCs), nicotinamide mononucleotide, and rapamycin to determine whether and how they responded to the antiaging interventions. The results showed that these treatments can significantly reduce the level of inflammatory factors (IL-6 and TNF-α). MSCs effectively improved the heart functions of aged dogs. Three key potential age-related genes (PYCR1, CCRL2, and TOX) were reversed by MSC treatment, two of which (CCRL2 and TOX) are implicated in immunity. Overall, we profiled the transcriptomic pattern of domestic dogs and revealed that they may be a good model of aging, especially in anti-inflammatory investigations.

Radicicol, a heat shock protein 90 inhibitor, inhibits differentiation and adipogenesis in 3T3-L1 preadipocytes.

Heat shock protein 90 (Hsp90) is involved in various cellular processes, such as cell proliferation, differentiation and apoptosis. As adipocyte differentiation plays a critical role in obesity development, the present study investigated the effect of an Hsp90 inhibitor radicicol on the differentiation of 3T3-L1 preadipocytes and potential mechanisms. The cells were treated with different concentrations of radicicol during the first 8days of cell differentiation. Adipogenesis, the expression of adipogenic transcriptional factors, differentiation makers and cell cycle were determined. It was found that radicicol dose-dependently decreased intracellular fat accumulation through down-regulating the expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT element binding protein α (C/EBPα), fatty acid synthase (FAS) and fatty acid-binding protein 4 (FABP4). Flow cytometry analysis revealed that radicicol blocked cell cycle at G1-S phase. Radicicol redcued the phosphorylation of Akt while showing no effect on ß-catenin expression. Radicicol decreased the phosphorylation of phosphoinositide-dependent kinase 1 (PDK1). The results suggest that radicicol inhibited 3T3-L1 preadipocyte differentiation through affecting the PDK1/Akt pathway and subsequent inhibition of mitotic clonal expansion and the expression/activity of adipogenic transcriptional factors and their downstream adipogenic proteins.

Involvement of Inheritance in Determining Telomere Length beyond Environmental and Lifestyle Factors.

All linear chromosomal ends have specific DNA-protein complexes called telomeres. Telomeres serve as a "molecular clock" to estimate the potential length of cell replication. Shortening of telomere length (TL) is associated with cellular senescence, aging, and various age-related diseases in humans. Here we reviewed the structure, function, and regulation of telomeres and the age-related diseases associated with telomere attrition. Among the various determinants of TL, we highlight the connection between TL and heredity to provide a new overview of genetic determinants for TL. Studies across multiple species have shown that maternal and paternal TL influence the TL of their offspring, and this may affect life span and their susceptibility to age-related diseases. Hence, we reviewed the linkage between TL and parental influences and the proposed mechanisms involved. More in-depth studies on the genetic mechanism for TL attrition are needed due to the potential application of this knowledge in human medicine to prevent premature frailty at its earliest stage, as well as promote health and longevity.

The Cross Talk between Cellular Senescence and Melanoma: From Molecular Pathogenesis to Target Therapies.

Melanoma is a malignant skin tumor that originates from melanocytes. The pathogenesis of melanoma involves a complex interaction that occurs between environmental factors, ultraviolet (UV)-light damage, and genetic alterations. UV light is the primary driver of the skin aging process and development of melanoma, which can induce reactive oxygen species (ROS) production and the presence of DNA damage in the cells, and results in cell senescence. As cellular senescence plays an important role in the relationship that exists between the skin aging process and the development of melanoma, the present study provides insight into the literature concerning the topic at present and discusses the relationship between skin aging and melanoma, including the mechanisms of cellular senescence that drive melanoma progression, the microenvironment in relation to skin aging and melanoma factors, and the therapeutics concerning melanoma. This review focuses on defining the role of cellular senescence in the process of melanoma carcinogenesis and discusses the targeting of senescent cells through therapeutic approaches, highlighting the areas that require more extensive research in the field.

A rationally designed fluorescence probe achieves highly specific and long-term detection of senescence in vitro and in vivo.

Senescent cells (SnCs) are implicated in aging and various age-related pathologies. Targeting SnCs can treat age-related diseases and extend health span. However, precisely tracking and visualizing of SnCs is still challenging, especially in in vivo environments. Here, we developed a near-infrared (NIR) fluorescent probe (XZ1208) that targets ß-galactosidase (ß-Gal), a well-accepted biomarker for cellular senescence. XZ1208 can be cleaved rapidly by ß-Gal and produces a strong fluorescence signal in SnCs. We demonstrated the high specificity and sensitivity of XZ1208 in labeling SnCs in naturally aged, total body irradiated (TBI), and progeroid mouse models. XZ1208 achieved a long-term duration of over 6 days in labeling senescence without causing significant toxicities and accurately detected the senolytic effects of ABT263 on eliminating SnCs. Furthermore, XZ1208 was applied to monitor SnCs accumulated in fibrotic diseases and skin wound healing models. Overall, we developed a tissue-infiltrating NIR probe and demonstrated its excellent performance in labeling SnCs in aging and senescence-associated disease models, indicating great potential for application in aging studies and diagnosis of senescence-associated diseases.

Longevity-Associated Transcription Factor ATF7 Promotes Healthspan by Suppressing Cellular Senescence and Systematic Inflammation.

Aging is characterized by persistent low-grade systematic inflammation, which is largely responsible for the occurrence of various age-associated diseases. We and others have previously reported that long-lived people (such as centenarians) can delay the onset of or even escape certain major age-related diseases. Here, by screening blood transcriptome and inflammatory profiles, we found that long-lived individuals had a relatively lower inflammation level (IL6, TNFα), accompanied by up-regulation of activating transcription factor 7 (ATF7). Interestingly, ATF7 expression was gradually reduced during cellular senescence. Loss of ATF7 induced cellular senescence, while overexpression delayed senescence progress and senescence-associated secretory phenotype (SASP) secretion. We showed that the anti-senescence effects of ATF7 were achieved by inhibiting nuclear factor kappa B (NF-κB) signaling and increasing histone H3K9 dimethylation (H3K9me2). In Caenorhabditis elegans, ATF7 overexpression significantly suppressed aging biomarkers and extended lifespan. Our findings suggest that ATF7 is a longevity-promoting factor that lowers cellular senescence and inflammation in long-lived individuals.

The calcium-sensing receptor promotes adipocyte differentiation and adipogenesis through PPARγ pathway.

Adipocyte differentiation and adipogenesis are closely related to obesity and obesity-induced metabolic disorders. The calcium-sensing receptor (CaSR) has been reported to play an antilipolytic role in human adipocyte and regulate cell differentiation in many tissues. However, the effects of CaSR on adipocyte differentiation and adipogenesis have not been clarified. In the study, we observed that activation of CaSR significantly promoted adipocyte differentiation and adipogenesis in human SW872 adipocytes. Gene expression analysis revealed that the CaSR activation increased the transcription factor proliferator-activated receptor γ (PPARγ) and its downstream genes including CCAAT element binding protein α (C/EBPα), adipose fatty acid-binding protein (aP2), and lipoprotein lipase. The activity of glycerol-3-phosphate dehydrogenase was also increased after the stimulation of CaSR. In addition, levels of cyclic AMP and calcium which have been shown to regulate PPARγ gene expression were significantly affected by the activation of CaSR. These effects could be suppressed by CaSR small interfering RNA (CaSR-siRNA). In conclusion, our findings suggest that activation of CaSR promotes differentiation and adipogenesis in adipocytes, which might be achieved by upregulating PPARγ and its downstream gene expressions. Therefore, CaSR in adipocytes may be involved in the pathogenesis of obesity by promoting adipocyte differentiation and adipogenesis.

Impact of Diets on Response to Immune Checkpoint Inhibitors (ICIs) Therapy against Tumors.

Immunotherapy has revolutionized the established therapeutics against tumors. As the major immunotherapy approach, immune checkpoint inhibitors (ICIs) achieved remarkable success in the treatment of malignancies. However, the clinical gains are far from universal and durable, because of the primary and secondary resistance of tumors to the therapy, or side effects induced by ICIs. There is an urgent need to find safe combinatorial strategies that enhance the response of ICIs for tumor treatment. Diets have an excellent safety profile and have been shown to play pleiotropic roles in tumor prevention, growth, invasion, and metastasis. Accumulating evidence suggests that dietary regimens bolster not only the tolerability but also the efficacy of tumor immunotherapy. In this review, we discussed the mechanisms by which tumor cells evade immune surveillance, focusing on describing the intrinsic and extrinsic mechanisms of resistance to ICIs. We also summarized the impacts of different diets and/or nutrients on the response to ICIs therapy. Combinatory treatments of ICIs therapy with optimized diet regimens own great potential to enhance the efficacy and durable response of ICIs against tumors, which should be routinely considered in clinical settings.

Specific Gain and Loss of Co-Expression Modules in Long-Lived Individuals Indicate a Role of circRNAs in Human Longevity.

Deep RNA sequencing of 164 blood samples collected from long-lived families was performed to investigate the expression patterns of circular RNAs (circRNAs). Unlike that observed in previous studies, circRNA expression in long-lived elderly individuals (98.3 ± 3.4 year) did not exhibit an age-accumulating pattern. Based on weighted circRNA co-expression network analysis, we found that longevous elders specifically gained eight but lost seven conserved circRNA-circRNA co-expression modules (c-CCMs) compared with normal elder controls (spouses of offspring of long-lived individuals, age = 59.3 ± 5.8 year). Further analysis showed that these modules were associated with healthy aging-related pathways. These results together suggest an important role of circRNAs in regulating human lifespan extension.

Telomere length is maternally inherited and associated with lipid metabolism in Chinese population.

Telomere is a unique DNA-protein complex which covers the ends of chromosomes to avoid end fusion and maintain the stability and integrity of chromosomes. Telomere length (TL) shortening has been linked to aging and various age-related diseases in humans. Here we recruited a total of 1031 Chinese individuals aged between 12 and 111 years, including 108 families with parents and their offspring. DNA was extracted from peripheral white blood cells and TL was measured by quantitative PCR (qPCR). We explored the associations of TL with age, gender and clinical variables, and tested the parental effects on TL variation. First, we found that TL was shortened with age, however, TL was better maintained in females than males. Second, there was a robust association of TL between mother and offspring, but not between father and their offspring. In addition, TL was inversely associated with visceral fat index in females, and positively associated with apolipoprotein A levels. Knockdown of the key genes for lipid metabolism (PNPLA2 and CPT1) shortened the TL in HepG2 cells. These findings indicate that TL is maternally inherited, and impairment of lipid metabolism may contribute to the TL shortening in the Chinese population.

Why Senescent Cells Are Resistant to Apoptosis: An Insight for Senolytic Development.

Cellular senescence is a process that leads to a state of irreversible cell growth arrest induced by a variety of intrinsic and extrinsic stresses. Senescent cells (SnCs) accumulate with age and have been implicated in various age-related diseases in part via expressing the senescence-associated secretory phenotype. Elimination of SnCs has the potential to delay aging, treat age-related diseases and extend healthspan. However, once cells becoming senescent, they are more resistant to apoptotic stimuli. Senolytics can selectively eliminate SnCs by targeting the SnC anti-apoptotic pathways (SCAPs). They have been developed as a novel pharmacological strategy to treat various age-related diseases. However, the heterogeneity of the SnCs indicates that SnCs depend on different proteins or pathways for their survival. Thus, a better understanding of the underlying mechanisms for apoptotic resistance of SnCs will provide new molecular targets for the development of cell-specific or broad-spectrum therapeutics to clear SnCs. In this review, we discussed the latest research progresses and challenge in senolytic development, described the significance of regulation of senescence and apoptosis in aging, and systematically summarized the SCAPs involved in the apoptotic resistance in SnCs.

Multiple time-series expression trajectories imply dynamic functional changes during cellular senescence.

Cellular senescence is a dynamic process driven by epigenetic and genetic changes. Although some transcriptomic signatures of senescent cells have been discovered, how these senescence-related signals change over time remains largely unclear. Here, we profiled the transcriptome dynamics of human dermal fibroblast (HDF) cells in successive stages of growth from proliferation to senescence. Based on time-series expression profile analysis, we discovered four trajectories (C1, C2, C3, C4) that are dynamically expressed as senescence progresses. While some genes were continuously up-regulated (C4) or down-regulated (C2) with aging, other genes did not change linearly with cell proliferation, but remained stable until entering the senescent state (C1, C3). Further analysis revealed that the four modes were enriched in different biological pathways, including regulation of cellular senescence. These findings provide a new perspective on understanding the dynamic regulatory mechanism of cellular senescence.

ETS1 acts as a regulator of human healthy aging via decreasing ribosomal activity.

Adaptation to reduced energy production during aging is a fundamental issue for maintaining healthspan or prolonging life span. Currently, however, the underlying mechanism in long-lived people remains poorly understood. Here, we analyzed transcriptomes of 185 long-lived individuals (LLIs) and 86 spouses of their children from two independent Chinese longevity cohorts and found that the ribosome pathway was significantly down-regulated in LLIs. We found that the down-regulation is likely controlled by ETS1 (ETS proto-oncogene 1), a transcription factor down-regulated in LLIs and positively coexpressed with most ribosomal protein genes (RPGs). Functional assays showed that ETS1 can bind to RPG promoters, while ETS1 knockdown reduces RPG expression and alleviates cellular senescence in human dermal fibroblast (HDF) and embryonic lung fibroblast (IMR-90) cells. As protein synthesis/turnover in ribosomes is an energy-intensive cellular process, the decline in ribosomal biogenesis governed by ETS1 in certain female LLIs may serve as an alternative mechanism to achieve energy-saving and healthy aging.

Involvement of calcium-sensing receptor in inhibition of lipolysis through intracellular cAMP and calcium pathways in human adipocytes.

The calcium-sensing receptor (CaSR) was cloned initially from the bovine parathyroid and its primary physiological role is maintaining constant blood Ca(2+) levels. Subsequently, CaSR was found to be expressed in human adipose tissue, however, its physiological functions remain unclear. In this study, the effect of CaSR on lipolysis and the mechanisms by which it functions were explored in SW872 cells. The results showed an inhibitory effect of CaSR on lipolysis after its being activated by GdCl(3), a CaSR agonist. CaSR stimulation decreased both cyclic AMP (cAMP) level and cAMP-dependent protein kinase A (PKA) activity. GdCl(3) treatment led to an increase in intracellular calcium ([Ca(2+)](i)) and mRNA level of phosphodiesterase3B (PDE3B). Furthermore, the downstream key enzymes of lipolysis, HSL and ATGL, were downregulated at both the transcription and translation levels by treatment with GdCl(3). Compared to the control group, the above effects were prevented by either NPS2390, a CaSR antagonist, or CaSR gene silencing by small interfering RNA (siRNA). These findings suggest that CaSR plays an antilipolytic role by mediating potential [Ca(2+)](i) and cAMP pathways and resultant downregulation of lipolysis key enzymes in adipocytes.

The calcium-sensing receptor affects fat accumulation via effects on antilipolytic pathways in adipose tissue of rats fed low-calcium diets.

Low-calcium intake is associated with increased risk of obesity, but the mechanism underlying this is not clear. We previously reported that the calcium-sensing receptor (CaSR) plays an important role in modulating the expression of rate-limiting lipolysis enzymes in human adipocytes. In the present study, rats were fed diets containing normal [0.50% (NC)], low [0.30% (LC)], or very low [0.15% (VLC)] calcium for 15 wk. Ten rats of each group were killed at wk 5, 10, and 15 of the intervention. The LC-fed rats had greater visceral fat mass, lower serum FFA and glycerol concentrations, and greater CaSR expression in white adipose tissue than did those fed the NC diet at wk 10 and 15. Hormone-sensitive lipase (HSL) and adipose TG lipase (ATGL) protein levels were lower, whereas fatty acid synthase mRNA in white adipose tissue was greater in the LC-fed rats compared with the NC-fed rats. These differences from the NC group were greater in the VLC group than in the LC group at wk 15. In vitro experiments showed that 1,25-dihydroxycholecalciferol stimulated the expression of CaSR through the nuclear vitamin D receptor (nVDR). This resulted in an antilipolytic effect by increasing intracellular calcium, decreasing the intracellular cAMP level, and downregulating HSL and ATGL protein expression in adipocytes. These effects were suppressed by either nVDR or CaSR small-interfering RNA. These results suggest that CaSR affects fat accumulation by mediating antilipolytic pathways in adipose tissue of rats fed low-calcium diets.