Senescent EC impairs adipocyte functions through SASP

Aging causes impaired metabolic homeostasis1,2,24, and the AT contains highly developed vascular networks that play an important role in maintaining adipocyte functions18,21,22. We, therefore, hypothesized that EC senescence might directly cause the adipocyte dysfunction and lead to age-related metabolic disorders. To analyze the role of EC senescence, we prepared two types of senescent EC: replicative senescent and premature senescent ECs by using human umbilical vein ECs. Premature senescence was induced by overexpressing the dominant negative (DN) form of the telomeric repeat-binding factor 2 (TERF2)25,26 in ECs. Their senescent phenotypes were validated through reduced proliferation, DNA damage, senescence-associated (SA) heterochromatin foci, reduced histone H3 lysine 9 dimethylation, SA β-galactosidase (Gal) activity, and increased CDK inhibitor and SASP factor expression (Supplementary Fig. 1a–g). We also confirmed that overexpression of TERF2DN did not induce EC apoptosis in the absence of a cytotoxic stress (Supplementary Fig. 1h). Replicative senescence of ECs was also validated through DNA damage, SA heterochromatin foci, and increased SASP factor expression (Supplementary Fig. 2) in addition to the reduced proliferation and increased CDK inhibitor expression, which we reported previously26. When treated with replicative senescent EC-conditioned medium (CM) that is enriched with endothelial senescence-messaging secretomes, 3T3-L1 adipocytes exhibited multiple senescence-like features such as SA-β-Gal activity, and increased CDK inhibitor and SASP factor expression compared with cells treated with control medium or CM derived from proliferating young EC (Fig. 1a–c). Although adipocytes are terminally differentiated nondividing cells, some of post-mitotic cells, including neurons and adipocytes, have been reported to exhibit several SA properties, and thus post-mitotic senescence-like state in terminally differentiated cells is an emerging concept27,28. Consistently, white adipose tissue (WAT) isolated from aged mice showed increased CDK inhibitor expression compared with that in WAT of young mice (Supplementary Fig. 3a). Of note, these 3T3-L1 adipocytes in senescence-like state showed impaired insulin signaling in association with reduced insulin receptor substrate (IRS)-1 expression (Fig. 1d–f). Similar results were obtained when 3T3-L1 adipocytes were treated with CM derived from premature senescent EC (Fig. 2). In contrast, treatment with senescent EC-CM did not induce such senescence features in 3T3-L1 preadipocytes or C2C12 myotubes (Supplementary Fig. 3b–d). In addition, we explored the paracrine effects of endothelial SASP on EC. Treatment with senescent EC-CM did not induce senescence features in young proliferating EC (Supplementary Fig. 4). These data suggest a higher susceptibility of mature adipocytes toward the endothelial SASP.

Fig. 1: Replicative senescent EC impairs adipocyte function through the SASP. a SA-β-Gal staining of 3T3-L1 adipocytes treated with the conditioned medium (CM) derived from proliferating young or replicative senescent EC. b CDK inhibitor expression in 3T3-L1 adipocytes treated with the control medium, or CM derived from proliferating young or replicative senescent EC (n = 8 biologically independent samples for control medium group; n = 6 biologically independent samples for EC-CM groups). c SASP factor expression in 3T3-L1 adipocytes treated with the control medium, or CM derived from proliferating young or replicative senescent EC (n = 4 biologically independent samples each). d Immunoblotting for the insulin signal pathway in 3T3-L1 adipocytes treated with the control medium (control), CM derived from proliferating young EC (young), or CM derived from replicative senescent EC (aged) in the presence or absence of insulin treatment. e Quantitative analysis for IRβ and Akt activation in response to insulin (n = 3 biologically independent samples each). f Immunoblotting for IRS-1 and IRS-2 in 3T3-L1 adipocytes treated with the control medium, or indicated EC-CM. Quantitative analysis was also shown (n = 3 biologically independent samples each). Non-repeated ANOVA with post hoc analysis of Fisher’s PLSD was used for difference evaluation between the groups (b, c, e, f). Data are presented as mean ± s.e. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Bars: 100 μm. Source data are provided as a Source Data file. Full size image

Fig. 2: Premature senescent EC impairs adipocyte function through the SASP. a SA-β-Gal staining in 3T3-L1 adipocytes treated with CM derived from control ECs (EC/GFP-CM) or premature senescent ECs (EC/TERF2DN-CM). b SPiDER-β-Gal staining in 3T3-L1 adipocytes treated with CM derived from control ECs (EC/GFP-CM) or premature senescent ECs (EC/TERF2DN-CM). c CDK inhibitor expression in 3T3-L1 adipocytes treated with the control medium, EC/GFP-CM or EC/TERF2DN-CM (n = 6 biologically independent samples each). d Immunoblotting for the insulin signal pathway and IRS-1 in insulin-stimulated 3T3-L1 adipocytes treated with the control medium, EC/GFP-CM or EC/TERF2DN-CM. Non-repeated ANOVA with post hoc analysis of Fisher’s PLSD was used for statistical analysis. Data are presented as mean ± s.e. *P < 0.05, **P < 0.01, and ****P < 0.0001. Bars: 100 μm. Source data are provided as a Source Data file. Full size image

Senescent EC induces oxidative stress in adipocytes

Oxidative stress is closely associated with cellular senescence. We found that treatment with senescent EC-CM caused excessive superoxide production in 3T3-L1 adipocytes, suggesting that endothelial senescence-messaging secretomes induced oxidative stress (Fig. 3a, b). Of note, treatment with the antioxidant N-acetylcystein (NAC) prevented endothelial SASP-mediated senescence features and impaired insulin signaling in adipocytes as well as β-nicotinamide mononucleotide, an intermediate of NAD+ biosynthesis that activates sirtuin29 (Fig. 3c–e). Pharmacological inhibition of ROS production pathways, such as xanthine and NADPH oxidase, failed to reverse senescence features in adipocytes treated with senescent EC-CM (Supplementary Fig. 5a), whereas expression of superoxide dismutase was reduced in these cells (Supplementary Fig. 5b). These data collectively suggest that senescent EC induces senescence-like state and impairs insulin signaling in adipocytes through the SASP by enhancing oxidative stress due to a defective redox state.

Fig. 3: Senescent EC impairs adipocyte functions by inducing oxidative stress. a Superoxide was detected (as red fluorescence) in 3T3-L1 adipocytes treated with the control medium, or CM derived from proliferating young or replicative senescent EC. b Superoxide was detected in 3T3-L1 adipocytes treated with the control medium, or CM derived from control ECs (EC/GFP-CM) or premature senescent ECs (EC/TERF2DN-CM). c CDK inhibitor expression in 3T3-L1 adipocytes treated with the indicated EC-CM in the presence or absence of βNMN or NAC (n = 4 biologically independent samples each). A two-tailed Student’s t test was used for difference evaluation between the two groups. Data are presented as mean ± s.e. *P < 0.05, **P < 0.01, and #not significant. d SA-β-Gal staining in 3T3-L1 adipocytes treated with the indicated EC-CM in the presence or absence of βNMN or NAC. e Immunoblotting for the insulin signal pathway and IRS-1 in insulin-stimulated 3T3-L1 adipocytes treated with the indicated EC-CM in the presence or absence of βNMN or NAC. Bars: 100 μm. Source data are provided as a Source Data file. Full size image

Generation of EC-specific progeroid mice

To analyze a role of senescent EC in age-related fat dysfunction in vivo, separately from effects of cellular senescence of other types of cells, we generated EC-specific progeroid mice that overexpress the DN form of TERF2 under the control of the Tie2 promoter (Tie2-TERF2DN-Tg). We obtained two lines of transgenic mice, both of which were viable and fertile. Senescence features, such as increased CDK inhibitor and SASP factor expressions, were detected in ECs isolated from Tie2-TERF2DN-Tg mice, while non-ECs did not show such features (Figs. 4a, b, Supplementary Fig. 6a, b). SA-β-Gal activity was also detected in ECs but not in non-ECs isolated from Tie2-TERF2DN-Tg mice (Fig. 4c). The percentage of SA-β-Gal-positive ECs in Tie2-TERF2DN-Tg mice was comparable with that in ECs isolated from naturally aged mice (Fig. 4d). We then explored whether these premature senescent ECs in Tie2-TERF2DN-Tg mice resemble senescent ECs in naturally aged mice. Principal component analysis for gene expression profiles assessed by DNA microarray revealed a significant similarity between ECs isolated from Tie2-TERF2DN-Tg and naturally aged mice, which significantly differed from ECs isolated from young mice (Fig. 4e). These data collectively indicate the EC-specific progeria in Tie2-TERF2DN-Tg mice, and provide a rationale for using this mouse model to analyze a role of EC senescence in aging.

Fig. 4: Generation of EC-specific progeroid mice. a CDK inhibitor expression in EC and non-EC isolated from the lung and WAT of WT or Tie2-TERF2DN-Tg mice (line #16) (n = 6 biologically independent samples for WT; n = 5 biologically independent samples for Tg). b SASP factor expression in EC and non-EC isolated from the WAT of WT or Tie2-TERF2DN-Tg mice (line #16) (n = 6 biologically independent samples each). c SA-β-Gal staining in EC and non-EC isolated from the lung of WT or Tie2-TERF2DN-Tg mice (line #16). SA-β-Gal-positive cells were counted (n = 5 biologically independent samples each). d EC and non-EC were isolated from the lung of young (7W) or aged (70W) mice. Cells were stained with SA-β-Gal, and staining-positive cells were counted (n = 6 biologically independent samples each). e Principalcomponent analysis for gene expression profiles assessed by DNA microarray in ECs isolated from the lung of young WT (7W), aged WT (70W), or Tie2-TERF2DN-Tg mice (20-week old). A two-tailed Student’s t test was used for difference evaluation between the two groups (a–d). Data are presented as mean ± s.e. *P < 0.05, **P < 0.01, and ****P < 0.0001. Bars: 100 μm. Source data are provided as a Source Data file. Full size image

EC senescence impairs systemic insulin sensitivity in vivo

Body weight was similar between the Tie2-TERF2DN-Tg and WT mice, although there was a trend toward an increase in the Tie2-TERF2DN-Tg mice (Fig. 5a). Body weight and body fat ratio at the age of 20-week old also showed a tendency toward an increase in the Tie2-TERF2DN-Tg mice, while the differences did not reach statistical significance (Fig. 5b, c). Of note, Tie2-TERF2DN-Tg mice exhibited impaired metabolic health at, as early as, 20-week old even while consuming normal chow (Fig. 5d, Supplementary Fig. 8a). Consistent with the reduced insulin sensitivity, serum insulin levels were higher in the Tie2-TERF2DN-Tg mice than those in the WT mice, whereas serum lipid profiles were similar across groups (Supplementary Figs. 7a and 8b). Because the insulin tolerance curve appeared to diverge especially at a later time point, we examined a possibly enhanced counter-regulatory response in the Tie2-TERF2DN-Tg mice through glucocorticoids. Serum corticosterone levels were similar between the WT and Tie2-TERF2DN-Tg mice (Supplementary Fig. 7b). On the other hand, CDK inhibitor expression increased not only in the EC-rich stromal vascular fraction (Supplementary Fig. 7c), but also in the mature adipocytes isolated from the WAT of Tie2-TERF2DN-Tg mice (Fig. 5e). Consistently, significant SA-β-Gal activity was detected in the WAT of Tie2-TERF2DN-Tg mice (Fig. 5f). In addition, some of inflammatory gene expression was increased in the WAT of Tie2-TERF2DN-Tg mice (Supplementary Fig. 8c). Furthermore, insulin signaling was impaired in the WAT of Tie2-TERF2DN-Tg mice in association with reduced IRS-1 expression as compared with that in WT mice (Fig. 5g, h). These results indicate that EC senescence is sufficient to induce senescence-like state and dysfunction in adipocytes in vivo, which probably have enough deleterious impact on systemic metabolic health. Notably, neither adipocyte senescence-like state nor metabolic disorders were observed in 10-week-old Tie2-TERF2DN-Tg mice (Fig. 6a, b), though ECs already showed senescence features at this age (Fig. 6c, d). These results indicate that WAT dysfunction and metabolic disorders in Tie2-TERF2DN-Tg mice occur postnatally after EC senescence, and are not due to developmental defects of the WAT. Blood vessel density in white and brown AT did not differ between the Tie2-TERF2DN-Tg and WT mice (Supplementary Fig. 8d, e). Similar phenotypes, including the EC senescence, impaired insulin sensitivity, and senescence-like state in WAT, were detected in an independent line of Tie2-TERF2DN-Tg mice at the age of 20 weeks old (Supplementary Fig. 9).

Fig. 5: EC-specific progeria impairs systemic metabolic health. a Body weight of WT or Tie2-TERF2DN-Tg (line #16) mice fed normal chow (NC) (n = 7 biologically independent animals for WT; n = 6 biologically independent animals for Tg). b, c Body weight (b) and body fat ratio (c) in WT or Tie2-TERF2DN-Tg mice fed NC at the age of 20 weeks old (n = 7 biologically independent animals for WT; n = 5 biologically independent animals for Tg). d Insulin tolerance test (ITT) in WT or Tie2-TERF2DN-Tg mice fed NC at the age of 20 weeks old (n = 7 biologically independent animals for WT; n = 6 biologically independent animals for Tg). e CDK inhibitor expression in mature adipocytes (MA) isolated from the WAT of WT or Tie2-TERF2DN-Tg mice (n = 6 biologically independent samples for WT; n = 5 biologically independent samples for Tg). f SA-β-Gal staining of subcutaneous (sWAT) or visceral epididymal WAT (eWAT) isolated from WT or Tie2-TERF2DN-Tg mice. g Immunoblotting for IRS-1 in the WAT isolated from WT or Tie2-TERF2DN-Tg mice (n = 7 biologically independent samples each). h Immunoblotting for the insulin signal pathway in the WAT, liver, and skeletal muscle of NC-fed WT or Tie2-TERF2DN-Tg mice with or without insulin treatment. A two-tailed Student’s t test was used for difference evaluation between the two groups (b–e, g). Data are presented as mean ± s.e. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Source data are provided as a Source Data file. Full size image

Fig. 6: EC-specific progeroid mice are metabolically healthy at the age of 10-week old. a ITT in WT or Tie2-TRF2DN-Tg mice fed normal chow at the age of 10-week old (n = 7 biologically independent animals for WT; n = 5 biologically independent animals for Tg). b CDK inhibitor expression in stromal vascular fraction (SVF) or mature adipocytes (MA) isolated from WAT of WT or Tie2-TRF2DN-Tg mice at the age of 10-week old (n = 10 biologically independent samples each). c CDK inhibitor expression in ECs isolated from lung (n = 15 biologically independent samples for WT; n = 10 biologically independent samples for Tg), and WAT (n = 13 biologically independent samples for WT; n = 10 biologically independent samples for Tg) of WT or Tie2-TRF2DN-Tg mice at the age of 10-week old. d SA-β-Gal staining in EC and non-EC isolated from the lung of WT or Tie2-TRF2DN-Tg mice at the age of 10-week old. Bars: 100 μm. SA-β-Gal-positive cells were counted (n = 6 independent fields each). A two-tailed Student’s t test was used for difference evaluation between the two groups (b–d). Data are presented as mean ± s.e. *P < 0.05, ***P < 0.001, and ****P < 0.0001. Source data are provided as a Source Data file. Full size image

WAT is highly susceptible to EC-mediated SASP

In contrast to WAT, there were no significant changes in CDK inhibitor expression in other tissues, including the liver, skeletal muscle, and BAT (Supplementary Fig. 10a–c). Gluconeogenic gene expression in the liver, PGC-1α expression in the skeletal muscle, thermogenic gene expression in the brown AT, and core temperature were not altered in Tie2-TERF2DN-Tg mice (Supplementary Fig. 10d–g). Moreover, insulin signaling was not impaired in the liver or skeletal muscle of Tie2-TERF2DN-Tg mice, in contrast to WAT (Fig. 5h). These data strongly suggest that WAT is more susceptible to the endothelial SASP than other organs as suggested by the in vitro studies.

Oxidative stress in WAT of EC-specific progeroid mice

WAT isolated from Tie2-TERF2DN-Tg mice showed significant oxidative DNA damage assessed by immunostaining for 8-hydroxy-2′-deoxyguanosine (8-OHdG) (Fig. 7a). We, therefore, explored whether enhanced oxidative stress plays a causative role in the impaired metabolic health in EC-specific progeroid mice. Administration of antioxidants for 10 weeks beginning at the age of 10 weeks old prevented the development of insulin resistance and WAT dysfunction in Tie2-TERF2DN-Tg mice (Fig. 7b–e). These data strongly suggest that oxidative stress induced by EC senescence plays a causative role in the WAT dysfunction and impaired systemic metabolic health in EC-specific progeroid mice.

Fig. 7: EC senescence impairs metabolic health by enhancing adipocyte oxidative stress. a Immunostaining for the 8-OHdG in the WAT of WT or Tie2-TERF2DN-Tg mice at the age of 20 weeks old. b ITT in WT or Tie2-TERF2DN-Tg mice with or without NAC treatment for 10 weeks beginning at 10-week old (n = 6 biologically independent animals for WT; n = 5 biologically independent animals for Tg). c CDK inhibitor expression in the WAT of control (vehicle-treated, n = 9 biologically independent samples for WT; n = 8 biologically independent samples for Tg) or NAC-treated (n = 5 biologically independent samples each) WT or Tie2-TERF2DN-Tg mice. Non-repeated ANOVA with post hoc analysis of Fisher’s PLSD was used for difference evaluation between the groups. d Immunostaining for the 8-OHdG in the WAT of WT or Tie2-TERF2DN-Tg mice treated with either vehicle or NAC. e Inflammatory gene expression in the WAT of control (vehicle-treated, n = 9 biologically independent samples for WT; n = 8 biologically independent samples for Tg) or NAC-treated (n = 5 biologically independent samples each) WT or Tie2-TERF2DN-Tg mice. A two-tailed Student’s t test was used for difference evaluation between the two groups (c, e). Data are presented as mean ± s.e. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and #not significant. Bars: 100 μm. Source data are provided as a Source Data file. Full size image

EC senescence is sufficient to impair metabolic health

To exclude the possible role of bone marrow (BM) cells that potentially express TERF2DN under the control of the Tie2 promoter, we generated BM chimeric mice (Supplementary Fig. 11). Tie2-TERF2DN-Tg mice transplanted with WT-BM cells remained to be insulin resistant, accompanied by the WAT senescence features, whereas WT mice transplanted with Tie2-TERF2DN-Tg-BM showed normal insulin sensitivity (Fig. 8a, b). These data indicate the minimal contribution of BM cells for the impaired metabolic health in Tie2-TERF2DN-Tg mice, and further validate a causative and crucial role of EC senescence in age-related metabolic disorders.

Fig. 8: EC senescence impairs metabolic health through the SASP. a ITT in recipient WT or Tie2-TERF2DN-Tg mice harboring BM of WT or Tg mice (n = 20 biologically independent animals for WT>WT group; n = 9 biologically independent animals for Tg>WT group; n = 9 biologically independent animals for WT>Tg group). b CDK inhibitor expression in the WAT isolated from recipient WT or Tie2-TERF2DN-Tg mice harboring BM of WT or Tg mice (n = 13 biologically independent samples for WT>WT group; n = 5 biologically independent samples for Tg>WT group; n = 6 biologically independent samples for WT>Tg group). c ITT in the recipient WT or Tie2-TERF2DN-Tg mice whose circulation was shared with WT (recipient–donor; WT–WT, n = 9 biologically independent animals) or Tie2-TERF2DN-Tg mice (n = 9 biologically independent animals for WT-Tg group; n = 6 biologically independent animals for Tg–Tg group). d ITT in the donor WT or Tie2-TERF2DN-Tg mice whose circulation was shared with WT (WT–WT, n = 12 biologically independent animals) or Tie2-TERF2DN-Tg mice (n = 7 biologically independent animals for WT-Tg group; n = 6 biologically independent animals for Tg–Tg group). Non-repeated ANOVA with post hoc analysis of Fisher’s PLSD was used for difference evaluation between the groups (a–d). Data are presented as mean ± s.e. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Source data are provided as a Source Data file. Full size image

EC progeria impairs metabolic health through the SASP

To investigate whether systemic metabolic disorders are induced by soluble factors in the blood circulation in EC-specific progeroid mice, we generated WT mice in which blood circulation was shared with Tie2-TERF2DN-Tg or littermate WT mice by using parabiosis procedure30. The surgical procedures for parabiosis were performed by using 10-week-old recipient WT and donor Tie2-TERF2DN-Tg or WT mice, and phenotypic analysis was performed after a 10-week period of shared circulation. Recipient WT mice whose circulation was shared with Tie2-TERF2DN-Tg mice (WT-Tg) showed significantly impaired insulin sensitivity compared with that in control recipient mice (WT–WT) to the extent similar to that in recipient Tg mice with shared circulation with Tg mice (Tg–Tg) (Fig. 8c). These data strongly suggest that senescence-messaging secretomes produced by senescent EC in blood circulation impair systemic metabolic health in an endocrine-dependent manner. We also explored whether blood from WT mice affects the metabolic disorders in Tie2-TERF2DN-Tg mice, and found that shared circulation with WT mice partly reversed the impaired insulin sensitivity in Tie2-TERF2DN-Tg mice (Fig. 8d).

IL-1a orchestrates the cytokine networks in senescent EC

Interleukin (IL)-1a has been reported as a marker of senescent but not quiescent ECs31. Furthermore, cell-surface-bound IL-1a has been reported to regulate senescence-associated cytokine networks in fibroblasts32. We also found that IL-1a showed a remarkable increase in replicative and premature senescent ECs in vitro, as well as in ECs isolated from Tie2-TERF2DN-Tg mice in vivo. Therefore, we explored a role of IL-1a in the endothelial SASP. Despite the high mRNA expression, IL-1a was hardly detectable in the culture medium of replicative senescent EC (Fig. 9a). It has been reported that inflammasome activation is needed to secrete IL-1a from cells, while membrane-bound IL-1a is also biologically active33,34. We, therefore, treated senescent EC with inflammasome activators, and found that IL-1a was abundantly secreted into the culture medium of replicative senescent EC with these stimuli (Fig. 9a). Moreover, FACS analysis revealed that the membrane-bound IL-1a increased in ECs isolated from naturally aged mice compared with that in ECs isolated from young mice (Fig. 9b). We then investigated the role of membrane-bound IL-1a in the senescence-messaging secretomes in EC. Treatment with IL-1 receptor antagonist abolished the increase of multiple cytokines in senescent EC compared with those in proliferating young EC (Fig. 9c). Notably, conditioned medium derived from senescent EC that was treated with IL-1 receptor antagonist failed to induce senescence-like state in 3T3-L1 adipocytes (Fig. 9d). Because IL-1 receptor antagonist inhibits both IL-1a and IL-1b, we examined the effect of IL-1a gene silencing by using short-interfering RNA. Similar to IL-1 receptor antagonist, gene silencing of IL-1a abrogated the increase of multiple cytokines in senescent EC compared with those in control EC, without affecting the CDK inhibitor expression (Fig. 9e, f). These data strongly suggest a critical role of membrane-bound IL-1a in the endothelial SASP by orchestrating the senescence-associated cytokine networks in EC.

Fig. 9: IL-1a orchestrates senescence-associated cytokine networks in EC. a IL-1a concentration in culture medium derived from young or replicative senescent EC in the presence or absence of inflammasome activators, such as monosodium urate crystal (MSU, 200 or 400 μg/mL) and adenosine triphosphate (ATP, 2.5 or 5 mM) (n = 4 biologically independent samples for young; n = 3 biologically independent samples for senescent EC). b FACS analysis of cell-surface IL-1a in ECs isolated from the lung of young (7W) or aged (70W) mice (n = 6 biologically independent samples each). A two-tailed Student’s t test was used for difference evaluation between the two groups. c SASP factor expression in young or replicative senescent EC in the presence or absence of IL-1 receptor antagonist (n = 6 biologically independent samples each). d CDK inhibitor expression in 3T3-L1 adipocytes treated with the conditioned medium derived from young or replicative senescent EC with or without IL-1 receptor antagonist treatment (n = 6 biologically independent samples each). e CDK inhibitor expression in young control EC (GFP), premature senescent EC (TERF2DN), or premature senescent EC transfected with IL-1a siRNA (n = 4 biologically independent samples each). f SASP factor expression in young control EC (GFP), premature senescent EC (TERF2DN), or premature senescent EC transfected with IL-1a siRNA (n = 4 biologically independent samples each). Non-repeated ANOVA with post hoc analysis of Fisher’s PLSD was used for difference evaluation between the groups (a, c–f). Data are presented as mean ± s.e. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and #not significant. Source data are provided as a Source Data file. Full size image

Analysis for EC secretomes

Characterization of changes in EC secretomes during aging provides invaluable information to identify SASP factors that mediate adipocyte dysfunction. We, therefore, performed the shotgun proteomics analysis using CM derived from young control and premature senescent EC. We detected 1307 proteins in the CM (Supplementary Excel sheet). Among these proteins, 380 proteins were differentially regulated (P < 0.1) in the senescent EC-CM compared with control EC-CM (Supplementary Fig. 12). Pathway analysis for the proteins that are increased in senescent EC-CM revealed several pathways that are potentially affected by the endothelial SASP, including signaling pathway for insulin-like growth factor, basic fibroblast growth factor platelet-derived growth factor, and extracellular matrix organization (Supplementary Fig. 13).