The REDD1–NF-κB–miRNAs–eNOS/SIRT1 axis mediates obesity-induced endothelial cell senescence and hypertension

Domain: biology
Published: 2026-03-12
Editorial outcome: major_revision

Abstract
Vascular dysfunction, including endothelial cell (EC) senescence and hypertension, is a hallmark of metabolic syndrome, yet its underlying mechanisms remain unclear. Here, we show that metabolic stress upregulates regulated in development and DNA damage response 1 (REDD1), driving vascular dysfunction. Overexpression of REDD1, but not the REDD1KK219/220AA mutant, which cannot activate atypical NF-κB, promotes EC senescence and hypertension through NSF-κB-dependent induction of miR-155-5p and miR-214-3p. These miRNAs suppress endothelial nitric oxide synthase (eNOS) and SIRT1 expression in human and mouse ECs. In obese male mice, REDD1 and miR-214-3p are upregulated, whereas eNOS and SIRT1 are downregulated, contributing to EC senescence, renal dysfunction, and hypertension. This phenotype is alleviated in mice lacking Redd1, EC-specific Redd1, or miR-214-3p, and in mice expressing Redd1KK219/220AA, but only partially by IKKβ inhibition. These findings identify a REDD1–atypical NF-κB–miRNAs–eNOS/SIRT1 axis as a critical mediator of obesity-induced vascular dysfunction and a promising therapeutic target.

Introduction
Obesity, which results from significant imbalances between caloric intake and energy expenditure, directly contributes to the development of metabolic and cardiovascular risk factors, including hypertension, dyslipidemia, type 2 diabetes, chronic inflammation, and elevated adipokines. These risk factors, in turn, lead to the onset of cardiovascular diseases (CVDs) and associated mortality. Obesity, particularly when accompanied by increased visceral adiposity, is a major cause of vascular dysfunction and hypertension, accounting for as much as 65–75% of the risk for primary hypertension, also known as essential or idiopathic hypertension1.

Endothelial cells (ECs), which form the inner layer (endothelium) of blood vessels, are critical for maintaining vascular homeostasis and blood pressure. They do so by modulating relaxation, angiogenesis, inflammation, and permeability through coordinated response mechanisms to multiple sensory inputs2. EC dysfunction has been identified as one of the inciting events responsible for CVDs, including hypertension and atherosclerosis1. Vascular dysfunction has been directly linked with EC senescence, which can be induced by several factors, particularly inflammation, oxidative stress, and obesity-induced metabolic changes3.

Cellular senescence is a state of irreversible cell-cycle arrest characterized by several hallmarks, including elevated levels of anti-proliferative proteins (e.g., p53, p21, and p16), accumulation of lysosomal senescence-associated β-galactosidase (SA-β-gal), and the induction of senescence-associated secretory phenotype (SASP, e.g., inflammatory cytokines and chemokines)4. Various intrinsic and extrinsic factors, such as DNA damage and oxidative stress, can trigger cellular senescence3. Obesity and metabolic abnormalities have also been reported as significant risk factors for EC senescence and vascular-associated CVDs, likely by downregulating anti-senescent genes, including endothelial nitric oxide synthase (eNOS) and SIRT15,6.

Nuclear factor-kappa B (NF-κB) is a critical factor in inflammatory responses, cellular senescence, and aging7. It is activated by factors associated with obesity and metabolic syndrome, including oxidized low-density lipoprotein (oxLDL), hyperlipidemia, and hyperglycemia8. Moreover, NF-κB stimulates the expression of various miRNAs (e.g., miR-31-5p, miR-155-5p, and miR-214-3p) that can negatively impact vascular eNOS and SIRT1 expression levels, leading to EC senescence, anti-angiogenesis, vasoconstriction, and hypertension5,9–12. Notably, the association between obesity and increased EC senescence and hypertension has been attributed to miRNA-mediated reductions in eNOS and SIRT1 levels or nitric oxide (NO) bioavailability in humans and rodents3,5,9. Therefore, inhibiting NF-κB pathways may maintain eNOS and SIRT1 expression levels, thereby preventing EC senescence and vascular dysfunction13-15. Furthermore, exogenous NO delivery and SIRT1 overexpression can mitigate EC senescence by increasing telomerase activity and preventing telomere shortening16,17. These findings suggest that obesity induces vascular dysfunction and consequent CVDs by suppressing eNOS and SIRT1 expression via the NF-κB–miRNA axis.

Regulated in development and DNA damage 1 (REDD1), also known as DNA damage-inducible transcript 4 protein (DDIT4), is an endogenous inhibitor of mechanistic target of rapamycin in complex 1 (mTORC1) and an atypical activator of NF-κB. Its genetic response is triggered by obesity, metabolic abnormalities, and factors associated with metabolic syndrome, making it a significant factor in adipogenesis, inflammation, type 2 diabetes, and EC dysfunction17. Recent studies have shown that REDD1 plays an important role in inducing metabolic inflammation in obese mice by activating the atypical NF-κB pathway by binding to and sequestering IκB without activating IKK, a key mediator in the canonical and non-canonical NF-κB pathways18–20. In addition, REDD1 expression is elevated in the brains of aged mice, senescent ECs, and doxorubicin-exposed cardiomyocytes, and its knockdown can prevent EC senescence21–23, suggesting its involvement in the pathogenesis of EC senescence. While these findings indicate that REDD1 may contribute to EC senescence associated with CVD, the mechanisms linking REDD1 with obesity-induced vascular dysfunction remain unclear.

In this study, we investigated the pathogenic role of REDD1 and the molecular mechanisms underlying its role in EC senescence and hypertension in cultured ECs and mouse models under obesity-related conditions. Our results showed that REDD1 plays a central role in the pathogenesis of obesity-induced EC senescence and hypertension by stimulating NF-κB-dependent miR-155-5p/214-3p expression, subsequently downregulating eNOS and SIRT1 expression levels.

Results
Metabolic factors promote REDD1-dependent EC senescence.
As reported previously20, male mice fed a high-fat diet (HFD) for 16 weeks to induce diet-induced obesity (DIO) exhibited increased body weight and elevated serum levels of metabolic syndrome-inducing factors (e.g., leptin, resistin, high glucose, cholesterol, oxLDL, triglycerides, and free fatty acids) compared with lean mice fed a chow diet (Supplementary Fig. 1a). REDD1 expression was markedly increased in the intact aorta of DIO mice but reduced after endothelial removal (Fig. 1a, b and Supplementary Fig. 1b), confirming endothelial-specific REDD1 expression. Immunostaining confirmed its predominant localization in aortic ECs (Fig. 1c). In cultured human aortic endothelial cells (HAECs), treatment with the metabolic risk factors, including palmitic acid, cholesterol, leptin, resistin, oxLDL, or high glucose, significantly induced REDD1 expression (Fig. 1d, e and Supplementary Fig. 1c). Treatment with palmitic acid, oxLDL, high glucose, or resistin promoted EC senescence, as evidenced by increased SA-β-gal⁺ cells and the senescence markers p53 and p21, whereas siRNA-mediated knockdown of REDD1 prevented these changes (Fig. 1f–h and Supplementary Fig. 1d, e). Given that REDD1 activates NF-B18, we investigated its role in EC senescence. IKKβ knockdown partially suppressed the increases in SA-β-gal activity induced by palmitic acid, high glucose, or resistin, with weaker inhibitory effects for high glucose or resistin than for palmitic acid; however, NF-κB p65 knockdown more effectively, though not completely, blocked SA-β-gal activity induced by these metabolic factors (Supplementary Fig. 1f).

Notably, TNF-α, an obesity-induced inflammatory cytokine, increased senescence markers without inducing REDD1 expression, and these effects were blocked by IKKβ knockdown, but not by REDD1 knockdown (Supplementary Fig. 1g). Similarly, IL-6 failed to induce REDD1 mRNA expression (Supplementary Fig. 1h). Replicative (passage-dependent) and oxidative (H₂O₂) stress also increased REDD1, p53, and p21 together with SA-β-gal activity (Supplementary Fig. 2a, b). These findings suggest that REDD1 mediates EC senescence in response to metabolic, replicative, and oxidative stress, but not to classical inflammatory NF-κB activators, such as TNF-α.

REDD1 induces EC senescence via NF-κB–SIRT1/eNOS axis.
Adenoviral overexpression of REDD1 suppressed the anti-senescence protein SIRT1, increased acetylated and nuclear p53, and upregulated p21 (Fig. 2a, b), resulting in elevated SA-β-gal+ cells (Fig. 2c and Supplementary Fig. 3a). REDD1 also induced SASP gene expression, such as IL1B, IL6, IL8, and CCL2 mRNAs (Fig. 2d), as well as EC-specific SASP markers, including intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) (Fig. 2e). Since REDD1 can atypically activate NF-κB20, which negatively regulates eNOS9,10, we examined its effects on the eNOS–NO axis and NF-κB activation. REDD1 overexpression reduced eNOS expression and NO production, which was abolished by the NOS-specific inhibitor NG-nitro-L-arginine methyl ester (L-NAME) (Fig. 2f, g). Notably, knockdown of NF-κB p65, but not IKKβ, inhibited REDD1-mediated NF-B promoter activity (Supplementary Fig. 3b, c) and attenuated REDD1-induced changes in eNOS, SIRT1, p53/p21, SA-β-gal activity, and IL-6 mRNA levels, although residual SA-β-gal activity and IL-6 mRNA levels remained significantly elevated (Fig. 2h, i and Supplementary Fig. 3d–f). Furthermore, REDD1-mediated EC senescence was effectively reversed by the NO donor DETA-NO and by SIRT1 overexpression, but not by its dominant-negative (DN) mutant (Fig. 2j and Supplementary Fig. 3g, h). These results suggest that REDD1 promotes EC senescence primarily via the IKKβ-independent NF-κB–SIRT1/eNOS pathway.

REDD1 downregulates eNOS and SIRT1 via NF-κB-responsive miRNAs.
Given that NF-κB-responsive miRNAs can downregulate eNOS and SIRT15,9,12, we evaluated their role in REDD1-mediated EC senescence. Knockdown of DICER, a key enzyme for miRNA biosynthesis, substantially reversed the REDD1-induced suppression of eNOS, SIRT1, and NO production, and the increase in intracellular NO was abolished by L-NAME treatment (Fig. 3a, b and Supplementary Fig. 4a, b). DICER knockdown also attenuated REDD1-mediated increases in SA-β-gal activity, miR-155-5p and miR-214-3p levels, and IL6 mRNA levels; however, SA-β-gal activity and IL6 mRNA levels remained partially but significantly elevated (Fig. 3c–e and Supplementary Fig. 4c). As both miRNAs are regulated by NF-κB activation9,11, REDD1-mediated increases in miR-155-5p and miR-214-3p levels were suppressed by knockdown of NF-κB p65, but not IKKβ (Supplementary Fig. 4d, e), suggesting that REDD1 promotes miR-155-5p and miR-214-3p biogenesis via atypical NF-κB activation. TargetScan predicted that these miRNAs target the 3′-UTRs of eNOS and SIRT1 in a species-specific manner (Supplementary Fig. 4f). We therefore assessed the effects of REDD1 on both genes in HAECs and mouse aortic endothelial cells (MAECs) using wild-type (WT) and miRNA target site-mutant 3′-UTR constructs (Supplementary Fig. 4g). Luciferase reporter assays confirmed that REDD1 inhibited NOS3 and SIRT1 3′-UTRs in a miRNA binding site-dependent and cell type-specific manner (Supplementary Fig. 4h, i). Treatment with miR-155-5p and miR-214-3p inhibitors reversed REDD1-induced eNOS/SIRT1 suppression, NO reduction, SA-β-gal activity, and IL6 mRNA upregulation, whereas their mimics reproduced the REDD1-like effects in HAECs and MAECs (Fig. 3f–i; Supplementary Figs. 4j, k, 5a–h). In contrast, REDD1KK219/220AA (REDD1KKAA), which cannot activate NF-κB19,20 but still binds TXNIP, a REDD1-interacting protein18 (Supplementary Fig. 6a) and failed to inhibit mTORC1 activity, as indicated by restored phosphorylation of S6K and 4E-BP1, did not significantly affect the levels of miR-155-5p, eNOS, SIRT1, senescence markers (SA-β-gal⁺ cells, p53, and p21), or SASP genes (IL1B and IL6) in HAECs, although some weak senescent features remained (Fig. 3j–m; Supplementary Fig. 6b, c). Notably, knockdown of RPTOR, which encodes Raptor, a key component of mTORC1, did not alter these outcomes, suggesting that REDD1-mediated inhibition mTORC1 is not sufficient to drive EC senescence. In addition, REDD1, but not REDD1KKAA, decreased the levels of phosphorylated Rb and cyclins D/E, without affecting cyclins A/B, consistent with G1-phase cell-cycle arrest associated with senescence (Supplementary Fig. 6d–g). We next examined the effects of obesity-related factors on NF-κB-responsive miR-155-5p and miR-214-3p expression. Palmitic acid and oxLDL increased these miRNAs in HAECs and MAECs, and this induction was effectively inhibited by NF-κB p65 knockdown and substantially reduced by REDD1 or IKKβ silencing, although residual miRNA levels remained significant (Supplementary Fig. 7a, b). These results suggest that REDD1, induced by obesity-related factors, promotes EC senescence through the atypical and canonical NF-κB-dependent miR-155-5p and miR-214-3p pathways, resulting in eNOS and SIRT1 downregulation.

REDD1, but not REDD1KKAA, induces vascular dysfunction in vivo.
To investigate the in vivo role of REDD1 in vascular dysfunction, male mice were intravenously injected with adenovirus encoding REDD1 (Ad-REDD1) or its mutant form (Ad-REDD1KKAA). In both groups, Redd1 mRNA levels were comparably upregulated across most tissues, showing the highest levels in the liver, markedly increased expression in the heart and aorta, and minimal expression in adipose tissue (Supplementary Fig. 8a). Serum levels of obesity-related factors, such as leptin, resistin, and triglycerides, showed no significant changes, whereas TNF-α was elevated only in Ad-REDD1-injected mice (Supplementary Fig. 8b). Unlike that of Ad-REDD1KKAA, systemic delivery of Ad-REDD1 increased SA-β-gal+ staining in the aorta, particularly in the aortic endothelium, and elevated miR-214-3p levels in mouse aortic tissues compared with Ad-Control (Fig. 4a, b). Similarly, Ad-REDD1 delivery reduced eNOS and SIRT1 expression while upregulating p53 and SASP markers, including ICAM-1 protein as well as Il1b, Il6, and Vcam1 mRNAs (Fig. 4c–e). Ad-REDD1, but not Ad-REDD1KKAA, decreased aortic NO and cGMP levels, impaired vasorelaxation of thoracic aortic and mesenteric arterial rings in response to acetylcholine (ACh), and increased systolic blood pressure (SBP), without affecting heart rate (Fig. 4f–h and Supplementary Fig. 8c). The role of eNOS in REDD1-mediated vascular senescence and hypertension was further evaluated using Nos3−/− mice. Compared with WT mice treated with Ad-control, Nos3−/− mice receiving Ad-control exhibited higher aortic SA-β-gal activity and p53 expression, without changes in SIRT1 levels. Upon Ad-REDD1 delivery in Nos3−/−mice, SIRT1 levels were reduced and p53 expression was markedly enhanced (Supplementary Fig. 8d, e). Although miR-214-3p increased after REDD1 overexpression in Nos3−/− mice, vascular NO and cGMP production remained unchanged, ACh-induced aortic vasorelaxation continued to be impaired, and SBP remained elevated (Supplementary Fig. 8f–i). In Nos2−/− mice, Ad-REDD1 delivery increased SA-β-gal activity and p53 expression, elevated vascular miR-214-3p, suppressed eNOS and SIRT1 expression, reduced vascular NO and cGMP production, impaired ACh-induced aortic vasorelaxation, and elevated SBP (Supplementary Fig. 8j–p). These results suggest that REDD1 overexpression induces vascular senescence and hypertension in vivo, accompanied by increased miR-214-3p levels and decreased eNOS and SIRT1 expression.

Redd1 loss attenuates DIO-induced vascular senescence and hypertension.
Given the critical role of REDD1 in metabolic disorders, including obesity and type 2 diabetes18,20, we assessed its contribution to vascular senescence and blood pressure in obese male mice. Compared with their lean counterparts, DIO-WT mice showed increased SA-β-gal+ staining in the aortas, especially in the endothelium, and this increase was reduced in DIO-Redd1−/− mice (Fig. 5a). Consistent with these findings, DIO elevated miR-214-3p expression in the aortas and serum of WT mice compared with that in lean controls, which was lower in DIO-Redd1−/− mice (Fig. 5b). Confocal imaging and biochemical analysis showed that DIO increased REDD1 levels and decreased eNOS and SIRT1 expression in the aortic endothelium of WT mice, accompanied by elevated total and acetylated p53, senescence markers (p53, p21, and p16), and SASP mRNAs (Il1b, Il6, and Vcam1) (Fig. 5c–g). These changes were attenuated in DIO- Redd1−/− mice, indicating that REDD1 plays a key role in DIO-induced vascular senescence. Moreover, DIO-WT mice demonstrated reduced aortic NO and cGMP production compared with their lean controls, and these levels were partially restored in DIO-Redd1−/− mice, with the recovery abolished by L-NAME (Fig. 5h). Consistently, ACh-mediated aortic vasorelaxation responses were impaired in DIO-WT aortas but improved in DIO-Redd1−/− mice (Fig. 5i). Likewise, SBP was higher in DIO-WT mice than in lean controls, and this effect was partially, but significantly, reduced in DIO-Redd1−/− mice (Fig. 5j). Notably, lean WT and Redd1−/− mice showed no significant differences, whereas DIO-Redd1−/− mice exhibited significantly impaired vascular function compared with lean Redd1−/− mice (Fig. 5b, e–j). These results indicate that REDD1 deficiency is associated with reduced obesity-induced EC senescence and hypertension, with partial reduction of miR-214-3p levels and partial restoration of eNOS, and SIRT1 expression, suggesting that REDD1 contributes to, but does not solely drive, these phenotypes.

More vascular protection by Redd1 deletion than IKK inhibition.
Given the established role of canonical NF-κB signaling in obesity-induced CVDs8, we compared the relative contributions of REDD1-mediated atypical NF-κB activation and IKK-dependent canonical NF-κB signaling in DIO-associated vascular dysfunction. Treatment with the IKK inhibitor Bay 11-7082 reduced vascular senescence and molecular alterations in both DIO-WT and DIO-Redd1−/− mice, as reflected by reduced SA-β-gal+ staining, suppression of miR-214 upregulation, partial restoration of eNOS and SIRT1, attenuation of p53 protein and Il6 and Vcam1 mRNA levels, and improved NO-cGMP signaling (Fig. 6a–e and Supplementary Fig. 9a). Bay 11-7082 also partially restored ACh-induced vasorelaxation in thoracic aortic and mesenteric arterial rings in both groups, an effect abolished by L-NAME, whereas the NO donor sodium nitroprusside induced comparable maximal relaxation across groups (Fig. 6f, g and Supplementary Fig. 9b). Moreover, Bay 11-7082 attenuated the DIO-induced elevation in SBP in both genotypes (Fig. 6h). Notably, the protective effects of Bay 11-7082 in DIO-WT mice were less pronounced than those observed in untreated DIO-Redd1−/− mice (Fig. 6a–f, h and Supplementary Fig. 9a, b), suggesting that the REDD1-mediated atypical NF-κB activation may play a more important role than the canonical NF-κB pathway in mediating obesity-induced vascular senescence and hypertension.

Redd1KKAA mice are protected from DIO-induced vascular dysfunction.
To delineate the role of atypical NF-κB activation by REDD1, we analyzed Redd1KKAA mice. Consistent with previous findings20, DIO increased circulating levels of leptin, resistin, glucose, cholesterol, triglycerides, free fatty acids, and TNF-α in WT mice, but these elevations were attenuated in Redd1KKAA mice, with no significant differences observed between genotypes under lean conditions (Supplementary Fig. 10a). Under DIO conditions, Redd1KKAA mice showed reduced SA-β-gal+ staining in the aortic endothelium (Fig. 7a) and were protected from the DIO-induced miR-214-3p upregulation in both aortic and serum measurements (Fig. 7b). Redd1KKAA mice maintained eNOS and SIRT1 expression and showed reduced senescence and SASP markers (p53, p21, p16, Il1a, Il1b, Il6, and Vcam1) under DIO conditions (Fig. 7c–e), leading to improved NO and cGMP production, enhanced ACh-induced aortic vasorelaxation, and attenuated SBP elevation (Fig. 7f–h). In contrast, lean mice showed no such changes (Fig. 7a–h). These results suggest that REDD1 promotes obesity-induced EC senescence and hypertension primarily through the atypical NF-κB–miR-214-3p–eNOS/SIRT1 axis, while also modulating systemic metabolic parameters as reported previously20.

Redd1ΔEC mice are protected from DIO-induced vascular dysfunction.
To define the role of endothelial REDD1, we examined Redd1ΔEC mice. DIO increased the serum levels of obesity-associated and inflammatory factors similarly in WT and Redd1ΔEC mice, with no statistically significant differences between genotypes (Supplementary Fig. 10b). Nonetheless, DIO-induced SA-β-gal+ staining in the aortic endothelium was attenuated in Redd1ΔEC mice (Fig. 8a). Endothelial deletion of REDD1 also mitigated aortic miR-214-3p induction, although the corresponding serum effect was less pronounced (Fig. 8b). Consequently, eNOS and SIRT1 expression, which were reduced in DIO-Redd1fl/fl mice, were partially preserved in DIO-Redd1ΔEC mice (Fig. 8c). Similarly, senescence-associated markers (p53, acetylated p53, p21, and p16) and SASPs (Il1a, Il1b, Il6, and Vcam1) were also attenuated in DIO-Redd1ΔEC mice (Fig. 8c–e). Consistent with these findings, Redd1ΔEC mice exhibited improved vascular NO and cGMP production, ACh-induced aortic vasorelaxation, and SBP under DIO conditions (Fig. 8f–h). These protective effects were not observed in lean controls (Fig. 8a–h). To assess the therapeutic relevance of endothelial Redd1 deletion, post-obesity EC-specific (DIO-Redd1ΔEC-post) mice were generated after confirming obesity by serum leptin and triglyceride measurements following 14 weeks of HFD feeding (Supplementary Fig. 11a, b). Two weeks later, assessment revealed significant improvements in vascular senescence, ACh-induced mesenteric arterial vasorelaxation, blood pressure, and related molecular markers compared with DIO-Redd1fl/fl controls (Supplementary Fig. 11c–i), closely resembling the protective effects observed in DIO-Redd1ΔEC mice. These results suggest that endothelial REDD1 is an important mediator of DIO-induced vascular senescence and hypertension, independent of systemic metabolic alterations.

miR-214-3p deficiency alleviates DIO-induced vascular dysfunction.
To determine the role of miR-214-3p in vascular dysfunction, we used miR-214-3p−/− mice. HFD feeding similarly increased obesity-associated serum factors in WT and miR-214-3p−/− mice, with no significant differences between genotypes (Supplementary Fig. 10c). However, SA-β-gal+ staining in the aortic endothelium was markedly attenuated in DIO-miR-214-3p−/− mice compared with that in DIO-WT mice, whereas no significant differences were observed under lean conditions (Fig. 9a). MiR-214-3p was upregulated in the aorta and serum of DIO-WT mice, but remained undetectable in miR-214-3p−/− mice (Fig. 9b). Under DIO conditions, REDD1 expression increased similarly in the aortas of both genotypes; however, reductions of eNOS and SIRT1 and elevations of p53, p21, and p16 were observed only in DIO-WT mice (Fig. 9c). Thus, DIO-induced elevations in Il1a, Il1b, Il6, and Vcam1 were attenuated in miR-214-3p−/− mice (Fig. 9d, e). Furthermore, miR-214-3p deletion partially restored DIO-mediated reductions in vascular NO−cGMP signaling and ACh-induced aortic vasorelaxation of thoracic aortic rings (Fig. 9f, g), thereby ameliorating SBP elevation (Fig. 9h). These results suggest that miR-214-3p contributes to obesity-induced vascular dysfunction in DIO mice, including EC senescence and hypertension, at least in part through modulation of eNOS and SIRT1 expression.

Redd1 and miR-214-3p deletion mitigates DIO-induced renal dysfunction.
Given the crucial role of the renal vasculature in blood pressure regulation, we assessed whether the REDD1-miR-214 axis contributes to renal dysfunction under DIO conditions. In WT, DIO caused glomerulomegaly, renal fibrosis, and elevated serum creatinine, whereas these pathological changes were effectively attenuated in Redd1−/−, Redd1ΔEC, and miR-214-3p−/− mice (Fig. 10a–e and Supplementary Fig. 12). These mice also exhibited partial preservation of eNOS expression under DIO conditions (Fig. 10f). Collectively, these findings suggest that REDD1 contributes to DIO-induced renal dysfunction, a potential contributor to hypertension, via NF-κB-responsive miR-214-3p-mediated modulation of eNOS.

Methods
Cell culture and treatment
HAECs (#6100) and human aortic smooth muscle cells (HASMCs, #6110) were obtained from ScienCell Research Labs (San Diego, CA, USA). MAECs were obtained from iXcells Biotechnologies (#10MU-002, San Diego, CA, USA). ECs were cultured in EGM-2 medium (#CC-3156; Walkersville, MA, USA) supplemented with growth factors (#CC-4176; Lonza Bioscience) or EC growth medium (#MD-0010, iXcells Biotechnologies), whereas HASMCs were cultured in smooth muscle cell medium (#1101, ScienCell Research Labs). For studies involving replicative senescence, HAECs (1.5 × 104 cells/cm2) were cultured in M199 media (#11043023; Invitrogen, Carlsbad, CA, USA)10, and serially passaged using trypsinization with trypsin‐EDTA (#25200072, Gibco, Grand Island, NY, USA) when the cells reached approximately 80–90% confluence13. To determine premature senescence, ECs between passages 4 and 5 were transfected with siRNAs (80 nM, control siRNA, #sc-37007; human REDD1, #sc-45806; mouse Redd1, #sc-45807; human IKKβ, #sc-35644; human NF-κB p65, #sc-29410; mouse NF-κB p65, #sc-29411; human Raptor, #44069; human DICER, #sc40489; San Cruz Biotechnology), miRNA mimics or inhibitors (80 nM, Supplementary Table 1), pFLAG-REDD1 (1 µg DNA/ml)19, or plasmid DNAs (1 µg/ml; human WT or DN mutant H363Y SIRT1; Plasmid #1791, Addgene, Watertown, MA, USA) using Lipofectamine RNAiMAX or Lipofectamine 3 000 (Invitrogen). Cells were then treated with or without oxLDL (50 µg/ml), prepared by incubating native LDL (1.019 mg protein) with 10 µM CuSO4 for 4 h64, cholesterol (100 µM, #C8667; Sigma-Aldrich, St. Louis, MO, USA), glucose (25 mM, 25 mM L-glucose used for osmotic control), recombinant human resistin (100 ng/ml, #1359-RN; R&D systems, Minneapolis, MN, USA), recombinant human leptin (10 µg/ml, #398-LP; R&D Systems), recombinant human TNF-α (10 ng/ml, #10291-TA; R&D Systems), recombinant human IL-6 (40 ng/ml, #206-IL; R&D Systems), palmitate-BSA (300 µM), or H2O2 (50−150 µM) for 48 h. Palmitic acid-BSA solution (5 mM) was prepared by mixing 10 µL of palmitate solution (500 mM in ethanol, #P9767; Sigma-Aldrich) with 1 mL of BSA solution (10%, w/v in serum-free M199, #A8806; Sigma-Aldrich). Subsequently, the solution was filtered through a 0.22 µm pore size membrane filter (Millipore, Billerica, MA) and stored at −80°C until use. For some experiments, ECs were infected with control adenovirus (Ad-Ctrl) or adenoviral REDD1 (Ad-REDD1, 50−150 MOI).

Animal study
C57BL/6J (#000664: B6/J), Nos3−/− (#002684; B6.129P2-Nos3/J), and Nos2−/− (#002609; B6.129P2-Nos2tm1Lau/J) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). VE-cadherin-CreERT2 (#13073; C57BL/6-Tg(Cdh5-cre/ERT2)1Rha) mice were purchased from Taconic Biosciences (Rensselaer, NY, USA). Redd1−/−, Redd1KKAA, Redd1flox/flox (Redd1fl/fl), and miR-214-3p−/− mice were generated using CRISPR/Cas9 on a C57BL/6J genetic background20. Mice were housed in a pathogen-free animal facility (temperature, 18–24°C; humidity, 30–50%) with a 12:12 h light/dark cycle. Endothelial-specific Redd1-deficient (Redd1ΔEC) mice were generated by crossing Redd1fl/fl with VE-cadherin-CreERT2 mice and intraperitoneally administering 100 µL of tamoxifen solution (20 mg/ml stock solution in corn oil) once every 24 h for 5 consecutive days. Six- to eight-week-old male mice were randomized into two groups and fed either a chow diet (10 kcal% fat, #D12450B, Research diets/Joongah Bio, Suwon, Republic of Korea; lean mouse) or an HFD (60 kcal% fat, #D12492, Research diets/Joongah Bio; DIO mouse) for 16 weeks. To establish post-obesity EC-specific (DIO-Redd1ΔEC-post) mice, VE-cadherin-CreERT2:Redd1fl/fl mice were intraperitoneally injected with tamoxifen following 14 weeks of an HFD-induced DIO. To study the role of canonical NF-κB, DIO-WT and DIO- Redd1−/− mice were administered intraperitoneal injections of Bay 11-7082 (5 mg/kg, dissolved in 1% DMSO) or vehicle every other day for the last two weeks. In addition, 14-week-old male WT, Nos3−/−, and Nos2−/− mice were intravenously injected with Ad-C or Ad-REDD1 (1 × 109 plaque-forming units in a total volume of 10 µl/mice) and maintained for 12 days. Following anesthesia with an intraperitoneal injection of Avertin (200 mg/kg), blood samples were collected from the abdominal aorta, centrifuged at 3,000 × g for 10 min at 4°C, and immediately stored at −80°C. The aortas and mesenteric arteries were rapidly and carefully isolated to evaluate vascular senescence, vascular relaxation response, the eNOS/NO pathway, and the expression levels of various genes. All experiments were approved by the Animal Ethics Committee of Kangwon National University (approval numbers: KW-210630-1 and KW-210707-6) and were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (8th edition, NIH, USA).

SA-β-gal staining and immunohistochemistry
EC and aortic vessel senescence were assessed using the Senescence β-Galactosidase Staining Kit (#9860, Cell Signaling Technology, Danvers, MA, USA) according to the manufacturer’s protocol. Images of the stained ECs were captured using an Olympus IX71P phase-contrast inverted microscope, and the percentage of SA-β-gal+ senescent cells was quantified in three separate fields using ImageJ software (v1.53c, NIH). Mouse aortas were fixed, washed with ice-cold phosphate buffered saline (PBS), and incubated with β-gal staining solution. Images of the stained aortic vessels were captured using a digital camera. Aortic vessels were embedded in optimum cutting temperature (OCT) compound and sectioned every 8 µm from the middle portion of the descending thoracic aorta to the diaphragm using a cryostat microtome (HM525NX, Thermo Fisher Scientific, Waltham, MA, USA). Images of SA-β-gal-stained aortic sections were acquired using the Digital Slide Scanner (Ocus®40, Grundium Ltd., Finland). For immunohistochemistry, fresh aortic vessels embedded in OCT compound were sectioned at a thickness of 10 µm using a cryostat microtome. These sections were stained with an anti-CD31 antibody (1:100, #AF3628, RRID: AB_2161028; R&D Systems) and a donkey anti-goat IgG (H+L)-Alexa Fluor™ 647 (1:100, #ab150131, RRID: AB_2732857; Abcam, Boston, MA, USA) for EC analysis; an anti-α-SMA antibody (1:100, #A5228, RRID: AB_262054; Sigma-Aldrich) and a goat anti-mouse IgG (H+L)-Alexa Fluor™ 405 (1:100, #A-31553, RRID: AB_221604; Invitrogen) for vascular smooth muscle cell analysis; an anti-REDD1 antibody (1:100, #10638-1-AP, RRID: AB_2245711; Proteintech, Rosemont, IL, USA) and a goat anti-rabbit IgG (H+L)-Alexa Fluor™ 488 (1:100, #A-11006, RRID: AB_2576217; Invitrogen) for REDD1 expression analysis; and an anti-eNOS antibody (1:100, #PA1-037, RRID: AB_325774; Invitrogen) and goat anti-mouse IgG (H+L)-Alexa Fluor™ 405 (1:100, #A-31553, RRID: AB_221604; Invitrogen) for eNOS analysis. Fluorescence images of the slides were captured using a confocal laser scanning microscope (LSM-880, Carl Zeiss, Oberkochen, Germany).

Hematoxylin and eosin (H&E) and Masson’s Trichrome staining
Kidney tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned at a thickness of 5 µm using a microtome (Microm HM 340E, Thermo Scientific, Walldorf, Germany). Sections were then deparaffinized, rehydrated, and stained with H&E or a Trichrome stain kit (#ab150686, Abcam) according to the manufacturer’s instructions. Images were acquired using a digital slide scanner (Ocus®40, Grundium Ltd. Tampere, Finland), and glomerular size and fibrotic area were quantified in four randomly selected fields per section using the ImageJ software (NIH).

Measurement of glucose, cytokines, adipokines, and lipids
Blood glucose levels were measured using the Accu-Chek active blood glucose meter (Roche Diagnostics, Seoul, Republic of Korea). Plasma levels of leptin (#MOB00, R&D Systems), resistin (#MRSN00, R&D Systems), cholesterol (#BM-CDL-100, Biomax, Guri-si, Republic of Korea), triglyceride (#BM-TGR-100, Biomax), free fatty acid (BM-FFA-100, Biomax), and oxLDL (#MBS2512757, MyBiosource, San Diego, CA, USA) were analyzed using ELISA kits according to the manufacturers’ protocol.

Co-immunoprecipitation and Western blotting
Cells were suspended in RIPA buffer (ELPIS-Biotech Inc., Daejeon, Republic of Korea) and incubated on ice for 30 min for complete cell lysis19. Using a BioMasher-II homogenizer (Optima, Tokyo, Japan), aortic vessel tissues were homogenized in ice-cold protein extraction buffer containing 100 mM HEPES (pH 7.9), 10% glycerol, 5% Triton X-100, 250 mM NaF, 5 mM Na3VO4, and Halt Protease Inhibitor Cocktail (100×, Thermo Fisher Scientific)11. Lysates were centrifuged at 12,000 × g for 10 min at 4°C, and the supernatants were collected for analysis. Total protein extracts (1 mg) were precleared with protein G plus/protein A-agarose (#IP10; Millipore) for 1 h at 4°C and then subject to co-immunoprecipitation with a FLAG M2 antibody (#F1804, RRID: AB_262044; Sigma-Aldrich). The resulting immunoprecipitates and cell lysates (30−40 µg protein) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (#GE10600023; Cytiva, Marlborough, MA, USA). The membranes were blocked with 5% bovine serum albumin (BSA, #BSAS0.1; Bovogen Biologicals Pty Ltd., Australia) in Tris-buffered saline (50 mM Tris-Cl pH 7.5, 150 mM NaCl) containing 0.05% Tween-20 and analyzed using immunoblotting with the respective primary antibodies: ant-eNOS (1:1000, #PA1-037, RRID: AB_325774; Invitrogen), anti-DICER (1:1000, #5362, RRID: AB_2093073; Cell Signaling Technology), anti-IKKβ (1:1000, #2684, RRID: AB_2122298; Cell Signaling Technology), anti-REDD1 (1:1000, #10638-1-AP, RRID: AB_2245711; Proteintech), anti-p65 (1:1000, #8242T, RRID: AB_10859369; Cell Signaling Technology), anti-p53 (1:1000, #2524, RRID: AB_331743; Cell Signaling Technology), anti-acetyl-p53 (1:1000, #2570, RRID: AB_823591; Cell Signaling Technology), anti-p21 (1:1000, #SC-6246, RRID: AB_823591; Santa Cruz, Santa Cruz, CA, USA), anti-p16 (1:1000, #SC-1661, RRID: AB_628067; Santa Cruz), anti-SIRT1 (1:1000, #8469S, RRID: AB_10999470; Cell Signaling Technology), anti-ICAM-1 (1:1000, #MA5407, RRID: AB_223596; Invitrogen), anti-VCAM-1 (1:1000, #MA5-31965, RRID: AB_2809259; Invitrogen), anti-Raptor (1:1000, #2280, RRID: AB_561245; Cell Signaling Technology), anti-phospho-S6K (Thr389, 1:1000, #9234, RRID: AB_2269803; Cell Signaling Technology), anti-S6K (1:1000, #34475, RRID: AB_2943679; Cell Signaling Technology), anti-phospho-4E-BP1 (Thr37/46, 1:1000, #9459, RRID: AB_330985; Cell Signaling Technology), anti-4E-BP1 (1:1000, #9452, RRID: AB_331692; Cell Signaling Technology), anti--actin (1:5000, #A5441, RRID: AB_476744; Sigma-Aldrich), anti-TXNIP (1:1000, #14715, RRID: AB_2714178, Cell Signaling Technology), or anti--tubulin (1:10000, #2144S, RRID: AB_2210548; Sigma-Aldrich). The membranes were incubated with a HRP-conjugated goat anti-mouse (1:4000, #31430, RRID: APB_228307; Thermo-Fisher Scientific) or HRP-conjugated goat anti-rabbit IgG antibody (1:4000, #31460, RRID: AB_228341; Thermo-Fisher Scientific). The blots were visualized using an X-ray film and an enhanced chemiluminescence kit (#34580; Thermo Scientific), and protein band intensities were quantified using the ImageJ software (NIH).

FACS analysis
HAECs were infected with 150 MOI of Ad-Control, Ad-REDD1, and Ad-REDD1KKAA for 48 h. Cells were harvested using 1 ml of 0.25% trypsin-EDTA solution, quenched with culture medium containing 5% FBS, and centrifuged at 300  g for 5 min at room temperature. The cell pellet was washed, resuspended in PBS, and fixed with 4% paraformaldehyde for 10 min, followed by centrifugation at 1000  g for 5 min and resuspension at 4−6  106 cells/ml in cold PBS. Cells (500 µL) were fixed by gently vortexing with the dropwise addition of 5 ml of cold 70% ethanol and stored for at least 2 h at 4°C. Fixed cells were collected, resuspended in 500 µL of permeabilization solution [100 µl of permeabilization buffer (10, #00-8333-56, Invitrogen), 50 µl of propidium iodide (#P4864, Sigma-Aldrich), and 2.5 µl of RNase A solution (#70856, 1 ml, Merck, Darmstadt, Germany) in a total volume of 1 ml PBS], and incubated in the dark for 15 min. Flow cytometry data of the stained cells (10,000 cells/sample) were acquired using the BD FACSVerse Flow Cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using the FlowJo software, version 10.2 (BD Biosciences). The gating strategy for cell-cycle analysis is shown in Supplementary Fig. 14.

Reporter gene assay
To evaluate eNOS promoter activity, ECs were transfected with 1 µg/ml of pGL3-human eNOS promoter (1.6 kb)-firefly luciferase construct and 500 ng/ml of Renilla luciferase pGL4.74 vector using Lipofectamine 3000 (Invitrogen)10. To analyze target gene 3-UTR-reporter activity, cells were transfected with 2 µg/ml of the psiCHECK-2 vector containing either the WT or mutant 3-UTR of eNOS (human, 430 bp; mouse, 470 bp) or SIRT1 (human, 680 bp; mouse 720 bp) using Lipofectamine 3000 (Invitrogen). Following transfection for 4 h, the cells were recovered in fresh medium for 24 h, and reporter gene activity was assessed using the Dual-Luciferase Assay Kit (#E1960, Promega, Madison, WI, USA). Firefly luciferase activity was normalized to Renilla luciferase activity.

eNOS–NO–cGMP pathway analysis
In situ aortic NO production was determined in vessel segments using a fluorescence image-based method11. Vessels were cut longitudinally, pinned to the bottom of a Sylgard-coated chamber (with the endothelial layer on top), and incubated with 5 µM 4-amino-5-methylamino-2´,7´-difluorofluorescein diacetate (DAF-FM DA, #CAS 254109-22-3, Calbiochem) in HEPES buffer for 30 min at 37C in minimal light. The chamber allowed for static bath conditions during fluorescence measurements. After washing to remove excess DAF-FM DA, fluorescence images were acquired at every 30 s for 5 min using an Olympus BX51 epifluorescence microscope with optimized excitation and emission wavelength (470/525 nm), an intensified camera (658M-TL, Luca, Los Angeles, CA, USA), and the cellSens imaging software, ver.4.1 (Olympus, Hamburg, Germany). The average fluorescence intensity was calculated from five frames (2 × 2 binning) of the entire field of view using the Metamorph software (Olympus). Slope changes in fluorescence over time were determined by linear regression using the data analysis software Origin (version 7.5, OriginLab Corp, Northampton, MA, USA), wherein one arbitrary unit of NO production was defined as a 0.1 increase of DAF fluorescence intensity/s. Furthermore, intracellular NO levels were measured in ECs treated with the NO fluorescence probe DAF-FM DA using a confocal laser scanning microscope (LSM-880, Carl Zeiss, Oberkochen, Germany)65. To evaluate cGMP production, ECs (1 × 105 cells) were transfected with 80 nM of control or miRNA inhibitors using Lipofectamine 3000 (Invitrogen) and infected with 150 MOI of Ad-C or Ad-REDD1, followed by co-culturing with HASMCs (1 × 105 cells) for 24 h48. The co-cultured media and lysates of aortic tissues isolated from mice were collected to measure cGMP levels using an ELISA kit (#KGE003; R&D Systems). The protein concentration in total lysates obtained from cells and aortic tissues was determined using a BCA method.

Measurement of blood pressure and vascular relaxation
Blood pressure was measured using the non-invasive CODA-HT4 tail-cuff system (CODA, Kent Scientific, Torrington, CT, USA) according to the manufacturer’s protocol. In brief, each mouse was acclimated for at least 10 practice sessions for a minimum of 15 min, and blood pressure parameters were subsequently recorded. In each recording session, one set of five acclimation cycles followed by three sets of 10 acquisition cycles were performed. The average of the last 20 successful recordings was used for calculating the systolic and diastolic blood pressures and heart rates. The tail temperature was monitored before and during the measurement, and mice with a core temperature of 33–35°C were used. The vasorelaxation responses of isolated carotid and mesenteric arterial rings to acetylcholine (ACh) were assayed using a multiwire myography system (DMT-620M, Aarhus, Denmark)11. Arterial rings (≈ 2 mm in length) were mounted on tungsten wires in a myograph chamber filled with physiological salt solution maintained at 37°C and aerated with 95% O₂ and 5% CO₂. After equilibration and normalization to optimal resting tension, vessels were precontracted with phenylephrine (1 µM) to achieve approximately 70–80% of maximal contraction, followed by cumulative addition of ACh to generate concentration-response curves. Vasorelaxation was expressed as the percentage of relaxation relative to the precontracted tone.

Reverse transcription-quantitative PCR (RT-qPCR)
Total miRNAs were isolated from cells, tissues, and serum using the miRNeasy Mini kit (#217004; Qiagen, Hilden, Germany) or the miRNeasy serum/plasma kit (#217184; Qiagen). cDNA for miRNA quantification was synthesized from 1 µg of miRNA using the miScript II RT kit (#218161; Qiagen). MiRNA levels were then quantified via qPCR using target-specific primers (Supplementary Table 1)9. MiRNA levels were normalized to the housekeeping gene SNORD95. For mRNA quantification, total RNA was extracted from cultured cells and mouse tissues using the TRIzol reagent (#15596018, Invitrogen). First-strand cDNA was synthesized using M-MLV Reverse Transcriptase (#M1705; Promega), followed by qPCR for the quantification of target gene mRNA levels using target-specific primers (Supplementary Table 2). Expression levels of target gene mRNAs were normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase.

Statistics and reproducibility
Statistical analysis was performed using GraphPad Prism (version 9.0; GraphPad Software Inc.). No statistical methods were used to predetermine the sample size. The experiments were randomized, and investigators were blinded to group allocation during experiments and outcome analysis. No samples or animals were excluded from the analysis. For cell-based experiments, n represents the number of independent biological replicates, whereas for animal studies, n represents the number of mice per group, as indicated in the figure legends. All representative images were obtained from at least three independent experiments with similar results. Western blotting, SA-β-gal staining, intracellular NO measurements, and renal histological analyses were quantified using all independent replicates, with data shown in the main or Supplementary Figures. Non-quantified representative vascular images were obtained from at least three animals per group, and the corresponding numbers of biological replicates are specified in the figure legends. All values are presented as means ± standard error of the mean (s.e.m). Statistical significance was set at P < 0.05 and was determined using two-sided unpaired t-tests or two-sided multiple t-tests for comparisons between two groups or one-way or two-way ANOVA followed by the Holm–Sidak’s multiple comparisons test. Exact P values are provided in the figures wherever applicable.

Data availability
All data supporting the findings of this study are provided in the article, the Supplementary Information, and the Source Data file, including uncropped blots and the source data for all graphs in the main and supplementary figures. Additional raw data are available from the corresponding authors upon reasonable request.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors have investigated a new signaling axis involved in the regulation of endothelial senescence and the\ndevelopment of hypertension in a rodent model for diet-induced obesity (DIO). The authors show that overexpression of the\nstress response protein REDD1 is sufficient to cause atypical (IKK-independent) NF-κB activation in endothelial cells that\nleads to miR-214-3p (mouse) or miR155-5p (human)-dependent suppression of SIRT-1 and eNOS. They also show that\nREDD1-dependent suppression of eNOS and SIRT1 leads to EC senescence. Furthermore, studies presented in the\nmanuscript demonstrate that both EC-specific REDD1 knockout (or REDD1KKAA mutant) and miR-214-5p-/- mice do not\ndevelop EC senescence and hypertension phenotype after DIO. The authors conclude that targeting endothelial cell\nREDD1 could mitigate EC senescence and hypertension in rodent models of DIO. While the data are extensive and\ngenerally support the conclusions, numerous major concerns need to be addressed:\nMajor concerns:\n1) Support for atypical NFkB activation in the context of DIO is not provided by these studies. Obese mice exhibit robust TLR\nsignaling and canonical NFkB activation that is likely concurrent with REDD1-dependent atypical NFkB signaling. Cell\nstudies here that compare the effect of REDD1 overexpression with siRNAs targeting IKKB versus NFkB p65 do little to\nprovide insight into cardiovascular disease. While REDD1 may be sufficient to stimulate atypical NFkB signaling, that does\nnot imply the lack of a role in canonical NFkB signaling in the context of DIO. These pathways should also be carefully\nexamined in the transgenic mice with DIO.\n2) Fig. 2I supports that REDD1 overexpression modestly promotes SA-B-gal staining in cells with p65 knockdown. Please\nindicate if these two groups are significantly different. This modest increase occurs despite p65 knockdown efficiency that is\nnearly 100% (Sup. Fig. 2b). Inhibition of NFkB activity with p65 knockdown is consistent with the failure of REDD1\noverexpression to reduce eNOS/SIRT1 or increase p53/p21 in cells with sip65 (Fig. 2h). Together the data support an\nalternative REDD1-dependent pathway that also contributes to EC senescence (i.e., in addition to the p65-mediated\neNOS/SIRT1 pathway).\n3) REDD1-dependent IL-6 expression in Fig. 3c was prevented by miRNA inhibitors. This observation should be reconciled\nby the investigators, as it is not consistent with the working model (Fig. 3m/8i). NFkB directly binds to the IL6 promoter to\nenhance its transcription. Thus, REDD1-dependent NFkB activity should be independent of miRNAs and their regulation of\neNOS/SIRT. Related to this, the assessment of REDD1 protein in Fig. 3e/h is missing. One possibility is that REDD1\nexpression or other NFkB regulators are also influenced by miRNA inhibitor/activator, as these necessary controls are\nabsent.\n4) Positive controls are missing for the REDD1 KKAA mutant protein in Fig. 3, 4, and 6. While it is clear that the mutant does\nnot activate EC senescence, it should be demonstrated that it still acts to suppress other NFkB-independent downstream\neffectors of REDD1 (e.g. mTORC1). One possibility is that the mutant protein is simply misfolded. These controls would also\nhelp to ground the speculative statements included in the Discussion (lines 324-332).\n5) The manuscript provides no evidence for a causal role of eNOS or SIRT in diet-induced hypertension in mice. The only\ndata that goes beyond association is found in Fig. 2j, which supports its role in REDD1-mediated EC senescence and\nSupplemental Fig. 5. Authors should be careful not to overstate the findings related to eNOS in Supplemental Fig. 5, as the\nstudy was performed with healthy mice. Support for a causal role of SIRT1 in REDD1-mediated EC senescence and\nhypertension is lacking. Line 190-191: The speculatory clause regarding the potential role of SIRT1 is unnecessary. The title\nof Supplementary Fig. 3 also overstates the causal role, as only an association is demonstrated in the data. Also see line\n199.\n6) Figure 5: Care should be taken with the text to be sure that the data are not overstated. Dependence of REDD1 deficiency\non eNOS and SIRT1 in this figure is not demonstrated as suggested by figure’s title. Also, REDD1 deletion looks to partially\nrestore changes seen in the DIO model. It is not clear that statistical analysis was performed between lean and DIO REDD1-\n/- groups, many of which appear to be significantly different. This likely supports that REDD1 is a key driver of EC\nsenescence and hypertension, but not entirely responsible for the changes. Statistically significant differences between lean\nand DIO transgenic mice should also be indicated in other figures.\n7) In this manuscript the authors show that miR-214-3p loss alleviated DIO-induced vascular dysfunction. Based on prior\nliterature this might not be an endothelial-specific effect. miRNA-214-3p in the kidney contributes to the development of\nhypertension (PMCID: PMC6171279). Recent studies have also shown that upregulated miRNA-214 also modulates the\nsenescence of vascular smooth muscle cells in carotid artery stenosis (PMCID: PMC7227274). Importantly, miRNAs often\nhave a dual regulatory role. Studies have previously demonstrated that miRNA-214 214-3p protects against myocardial\nischemia-reperfusion injury (PMCID: PMC10066509) and knockdown of microRNA-214-3p promotes tumor growth and\nEpithelial-Mesenchymal Transition in prostate cancer (PMCID: PMC8656576). These are important considerations that\nshould must be addressed experimentally.\n8) A previous study has also demonstrated a role for REDD1 in doxorubicin-elicited endothelial cell dysfunction through\ndownregulation of VEGFR-2/3 expression (PMCID: PMC8568908). Inhibition of VEGF can induce cellular senescence\n(PMID: 21618508) can the authors speculate on this contrary observation.\n9) A previous study has reported that Kallistatin reduces vascular senescence and aging by regulating microRNA‐34a‐\nSIRT1 pathway. Another study has shown that reduced expression of microRNA-130a promotes endothelial cell\nsenescence (PMCID: PMC7346016). Since microRNA‐34a is upregulated (PMID: 22988100) and microRNA-130a is\nreduced (PMID: 23396142) in obese patients, the authors should try to rule out the role of other miRNAs in obesity-induced\nvascular senescence. Importantly, clinical evidence for increased miR-155-5p in obese patients would greatly enhance the\nimpact of this study.\n10) Figure 6: It is unclear if levels of obesity-related factors are similar in DIO WT versus REDD1 KYAA mice.\n11) The rigor of western blot analysis is poor with often only single replicates shown and no quantifications.\n12) While deletion of REDD1 from birth is a useful way to delineate pathways being regulated, it is not a therapeutically\ntranslational model as the disease is induced later in life. For the model to be therapeutically translational, the authors might\ntry to suppress REDD1 after the mice are made obese and DIO symptoms such as elevated serum parameters are\nobserved.\nMinor comments:\n1) SIRT1 is misspelled in the title.\n2) In the methods section the authors state the miR-214-5p-/- mice were generated (line 484). All the data shown in the\nmanuscript indicate that miR-214-3p-/- mice were used. This potentially calls into question all studies reported in the miR-\n214-3p-/- mice, but is hopefully just another typo.\n2) Many gene names in Supplementary Table 2 are incorrectly formatted. These include hyphens and a random use of\ncapitalization. REDD1 gene as Redd1, rather than DDIT4 is also unconventional.\n3) Line 130: suggest editing “effectively rescued REDD1-mediated promoter activity…”. The siRNA for p65 does not appear\nto rescue REDD1-dependent promoter activity, but rather is preventing it.\n4) Sup. Fig. 2c: suggest editing the confusing location of “ns” and bar between groups 2 and 4.\n5) Line 143: The REDD1-dependent increase in SA-B-gal was reduced but not “prevented”.\n6) Line 145: incorrect description. Potentially missing phrase “which was prevented by”…knockdown of p65.\n7) Line 290: “Despite being more readily detected at its mRNA level…” This statement is unclear. Are the authors implying a\nlack of coordination between mRNA and protein?\n8) The absence of cardiovascular deficits in diabetic REDD1-/- mice fed a high-fat diet has already been demonstrated and\nlinked to NFkB signaling (PMID: 38928166).", "reviewer_2": "In this manuscript, the authors investigated the function of REDD1 in regulating obesity-induced endothelia cell (EC) senescence and hypertension. They found that HFD feeding increased REDD1 expression in aortic endothelium in mice and obesity promote EC senescence in a REDD1-dependent manner. They further revealed that REDD1 plays a central role in the pathogenesis of obesity-induced EC senescence and hypertension by stimulating NF-κB-dependent miR-155-5p/214-3p expression, which leads to the downregulation of eNOS and SIRT1 expression. The manuscript included comprehensive in vitro and in vivo data. However, there are several issues with the mouse models the authors used and the described underlying mechanisms. I have the following specific comments:\n-The authors showed that systemic delivery of Ad-Redd1 but not Ad-Redd1KKAA induces EC senescence and hypertension. It is not clear whether Ad-Redd1 and Ad-Redd1KKAA can efficiently target other tissues in addition of aorta. What is the tissue distribution pattern of Redd1 or Redd1KKAA after delivery? REDD1overexpression has been shown to stimulate preadipocyte differentiation and proinflammatory cytokine expression through atypical IKK-independent NF-κB activation in adipose tissue (Nat Commun 13, 6303 (2022)). If Ad-Redd1 and Ad-Redd1KKAA delivery also led to increased Redd1 or Redd1KKAA expression in adipose tissue, this may affect the obesity and its’s associated phenotypes. The authors need to characterize the obesity and metabolic phenotypes carefully in those mice.\n-In Fig. 5, the authors showed Redd1 deficiency prevents DIO-induced EC senescence and hypertension. Again, did Redd1 deficiency affect the DIO directly? Obesity and metabolic phenotypes of those mice under chow and HFD feeding condition need to be characterized.\n-The authors generated EC-specific Redd1 mouse model which could address some of above concerns. However, the manuscript did not include basic characterization of these mice. Did deficiency of Redd1 in ECs protect mice from DIO, systemic inflammation, and other metabolic disorder. It is plausible that those factors can contribute indirectly to the observed EC senescence and hypertension phenotypes.\n- miR-155-5p and miR-214-3p have been well known to play diverse roles in regulating various gene expression and are also linked to cancer, inflammation, and many physiological processes. The authors should explore how the changed miR-155-5p and miR-214-3p affect the different gene/pathways in addition to eNOS and SIRT1. For example, RNA-seq experiments would give a better overview of the impact of miR-155-5p and miR-214-3p on EC gene expression. Do other genes/pathways contribute to observed EC senescence or dysfunction?", "reviewer_3": "In this study, authors analyzed the roles of REDD1 in mediating endothelial dysfunction due to obesity, and found that REDD1 promotes NF-kB signaling, which alters eNOS and SIRT1 via miR-214-3p. This mechanism is lengthy and possibly REDD1 can directly exert effects on endothelial cells considering that REDD1 is a stress protein known to induce senescence. There are several major problems:\n1. The evidence supporting the involvement of NF-kB signaling is weak. Also, NF-kB signaling is a generic inflammatory pathway which can induce many changes, not only miR-214-3p and others.\n2. REDD1 can induce cell senescence directly with mechanisms not only involving NF-kB signaling and eNOS.\n3. More evidence is needed in the involvement of eNOS and SIRT1.\n4. It remains unclear why REDD1 is activated in endothelial cells due to obesity. It might be possible that obesity-induced inflammation activates REDD1, which then induces endothelial senescence, instead of REDD1 inducing inflammation.\nA few additional comments:\nLine 3: SITR1 should be SIRT1\nLine 111: Please also test TNFa, a direct activator of NF-kB signaling.\nLine 171: There is insufficient evidence to support NF-kB involvement. It is likely that REDD1 induces senescence through other mechanisms.\nLine 189: The accelerated senescence due to Ad-Redd1 delivery in eNOS KO mice suggested that the proposed mechanism of eNOS might not be important. What is the relationship between eNOS and SIRT1? How could REDD1 regulate both of them? Any molecular links between eNOS and SIRT1?", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "This is a comprehensive and well-executed study identifying REDD1 as a central regulator of obesity-induced endothelial dysfunction and hypertension through an atypical NF-κB–miRNA–eNOS/SIRT1 axis. The authors demonstrate the molecular and phenotypic consequences of REDD1 activation using an elegant combination of in vitro endothelial models and multiple genetically modified mouse lines (Redd1⁻/⁻, Redd1ΔEC, Redd1KKAA, and miR-214-3p⁻/⁻). The work provides significant mechanistic insight into how metabolic stressors modulate vascular aging and function and has clear translational relevance to obesity-related cardiovascular disease.\nMajor Comments:\n1. Limited Vascular Phenotyping in Resistance Arteries: While the study thoroughly characterizes REDD1-mediated vascular changes in the aorta, this vessel is primarily a conduit artery and does not directly regulate systemic blood pressure. Since resistance arteries (e.g., mesenteric, renal arterioles) are key determinants of vascular tone and blood pressure, the absence of functional and molecular assessments in these vessels limits the physiological relevance of the findings. It would significantly strengthen the manuscript to include data on vasomotor responses, NO bioavailability, and eNOS expression/activity in resistance arteries from the same mouse models.\n2. Kidney Function and Pathology Not Evaluated: Given the central role of the renal vasculature in blood pressure regulation, the manuscript would benefit from analysis of renal function and histopathology. Data on glomerular structure, fibrosis, proteinuria, serum creatinine, and eNOS levels in renal tissue would offer critical insight into whether REDD1 influences hypertension through renal mechanisms in addition to vascular effects.\n3. Lack of Human Validation: While the preclinical models are robust, no data from human samples are presented to confirm the upregulation of REDD1 or its downstream effectors (miR-155-5p, miR-214-3p, eNOS, SIRT1) in obesity-related hypertension. Inclusion of data from human endothelial cells, vascular tissue, or patient serum (even observational) would enhance the translational relevance of the findings.\n4. Sex-Specific Differences Not Addressed: All animal experiments appear to have been conducted exclusively in male mice, which overlooks potential sex-specific differences in REDD1 expression, endothelial aging, and hypertensive responses. Including female cohorts or, at a minimum, discussing this limitation is recommended to improve the generalizability of the results.\nMinor Comments:\n1. DAF-FM DA as a Surrogate for NO Production: The authors use DAF-FM DA fluorescence to quantify NO production. While this is a common and convenient method, it measures bioavailable NO via reaction with NO-derived intermediates (e.g., N₂O₃) and is considered semi-quantitative. This technique does not directly assess NOS enzymatic activity, and the fluorescence signal may be influenced by oxidative stress and NO scavenging. To more definitively evaluate eNOS activity, the authors should consider, or at least acknowledge, alternative approaches such as [¹⁴C]-L-arginine to [¹⁴C]-L-citrulline conversion assays or EPR spectroscopy for detection of authentic NO\n2. Limitations of Tail-Cuff Blood Pressure Measurement: The authors report systolic, diastolic, and mean arterial pressures using a non-invasive tail-cuff system. While this method is suitable for SBP measurements, diastolic and MAP values are often unreliable due to methodological constraints. Suggested to remove DBP and thus mean BP in all figures. Telemetry or intra-arterial catheterisation is the gold standard for accurate, continuous blood pressure measurement, especially in studies assessing nuanced vascular phenotypes. The authors are encouraged to acknowledge this limitation in the discussion."}, "author_response": "Response to reviewers’ comments\nReviewer #1 (Remarks to the Author):\nThe authors have investigated a new signaling axis involved in the regulation of endothelial\nsenescence and the development of hypertension in a rodent model for diet-induced obesity\n(DIO). The authors show that overexpression of the stress response protein REDD1 is\nsufficient to cause atypical (IKK-independent) NF-κB activation in endothelial cells that\nleads to miR-214-3p (mouse) or miR155-5p (human)-dependent suppression of SIRT-1 and eNOS.\nThey also show that REDD1-dependent suppression of eNOS and SIRT1 leads to EC\nsenescence. Furthermore, studies presented in the manuscript demonstrate that both EC-\nspecific Redd1 knockout (or Redd1KKAA mutant) and miR-214-3p-/- mice do not develop EC\nsenescence and hypertension phenotype after DIO. The authors conclude that targeting\nendothelial cell REDD1 could mitigate EC senescence and hypertension in rodent models of\nDIO. While the data are extensive and generally support the conclusions, numerous major\nconcerns need to be addressed.\nMajor comments\nComment #1. Support for atypical NF-κB activation in the context of DIO is not provided by\nthese studies. Obese mice exhibit robust TLR signaling and canonical NF-ΚB activation that\nis likely concurrent with REDD1-dependent atypical NF-ΚB signaling. Cell studies here that\ncompare the effect of REDD1 overexpression with siRNAs targeting IKK versus NF-ΚB p65\ndo little to provide insight into cardiovascular disease. While REDD1 may be sufficient to\nstimulate atypical NF-κB signaling, that does not imply the lack of a role in canonical NF-κB\nsignaling in the context of DIO. These pathways should also be carefully examined in the\ntransgenic mice with DIO.\nResponse: (1) We thank the reviewer for this valuable comment. To address this, we\nexamined the effect of IKK inhibition on endothelial senescence and hypertension in DIO-WT\nand DIO-Redd1–/– mice. Mice were treated with the IKK inhibitor Bay 11-7082 (5 mg/kg, i.p.)\nevery other day during the final two weeks of HFD feeding.\n(2) Bay 11-7082 partially reduced vascular senescence, molecular alterations, and\nhypertension in both genotypes, including decreased SA-β-gal staining, suppressed miR-214\nupregulation, partial restoration of eNOS and SIRT1, reduced p53 protein, and lowered Il6 and\nVcam1 mRNA levels. These effects were less pronounced than those observed with Redd1\ndeletion (Fig. 6a–h; Supplementary Fig. 9a, b), indicating contributions of both atypical and\ncanonical NF-κB pathways, with greater protection upon Redd1 deletion (Lines 240–256).\n1\n(3) Protective effects were also observed, though incomplete, in Redd1KKAA mice, where\natypical NF-κB signaling is absent (Fig. 7). Palmitic acid and oxLDL increased these miRNAs\nin HAECs and MAECs, and this induction was effectively blocked by NF-κB p65 and\nsubstantially reduced by REDD1 or IKKβ knockdown, although residual miRNA levels\nremained significant (Supplementary Fig. 7a, b), which is consistent with the results of residual\nSA-β-gal activity in Fig. 1. These results suggest that both atypical and canonical NF-κB\npathways contribute to REDD1-mediated vascular dysfunction in DIO.\n(4) Together with cellular studies, these findings indicate that both atypical and canonical NF-\nκB signaling contribute to obesity-induced vascular dysfunction, with the REDD1–atypical NF-\nκB axis likely playing a more important role. Thus, this study provides evidence that REDD1\norchestrates endothelial senescence and vascular dysfunction primarily via atypical NF-κB\nactivation, while canonical signaling also contributes. Given the limited specificity of Bay 11-\n7082, further studies using EC-specific Ikkβ-deficient mice are needed to define the role of\ncanonical NF-κB signaling (Lines 350–357).\nComment #2. Fig. 2I supports that REDD1 overexpression modestly promotes SA-β-gal\nstaining in cells with p65 knockdown. Please indicate if these two groups are significantly\ndifferent. This modest increase occurs despite p65 knockdown efficiency that is nearly 100%\n(Sup. Fig. 2b). Inhibition of NF-ΚB activity with p65 knockdown is consistent with the failure of\nREDD1 overexpression to reduce eNOS/SIRT1 or increase p53/p21 in cells with sip65 (Fig.\n2h). Together the data support an alternative REDD1-dependent pathway that also contributes\nto EC senescence (i.e., in addition to the p65-mediated eNOS/SIRT1 pathway).\nResponse: (1) (1) As requested, we have indicated the statistical significance between the\nAd-Control and sip65 + Ad-REDD1 groups in Fig. 2I and Supplementary Fig. 3c, and ensured\nconsistent presentation across related figures.\n(2) We agree that, although modest, REDD1 can induce endothelial dysfunction independently\nof NF-κB. As described in the Discussion section (Lines 376–391), potential mechanisms\ninclude TXNIP-mediated oxidative stress, VEGFR-2/3 downregulation, and mitochondrial\ndysfunction, whereas mTOR inhibition is unlikely given its protective role in ECs. Our data also\nsuggest that the TXNIP pathway alone has only limited effects, as indicated by residual\nsenescence in Redd1KKAA (Fig. 3l), which retains TXNIP-binding activity (Supplementary Fig.\n6a). Thus, REDD1 functions as a critical regulator through the atypical NF-κB–eNOS/SIRT1\naxis, while NF-κB–independent pathways, such as TXNIP-mediated oxidative stress, VEGFR-\n2/3 and mitochondrial signaling, may also contribute modestly. Further studies are warranted\nto clarify their precise roles.\ninhibitors. This observation should be reconciled by the investigators, as it is not consistent\nwith the working model (Fig. 3m/8i). NF-κB directly binds to the IL6 promoter to enhance its\ntranscription. Thus, REDD1-dependent NF-κB activity should be independent of miRNAs and\ntheir regulation of eNOS/SIRT1. Related to this, the assessment of REDD1 protein in Fig. 3e/h\nis missing. One possibility is that REDD1 expression or other NF-κB regulators are also\ninfluenced by miRNA inhibitor/activator, as these necessary controls are absent.\nResponse: (1) We thank the Reviewer for highlighting the need to clarify the role of miRNAs\nand eNOS/SIRT1 in REDD1-mediated regulation of SASP-related cytokines, including IL-6.\n(2) Knockdown of NF-κB p65 or DICER reduced REDD1-induced miR-155-5p, miR-214-3p,\nSA-β-gal activity, and IL6 mRNA, although residual SA-β-gal and IL6 expression persisted (Fig.\n3c–e; Supplementary Fig. 4c–e). Similarly, IL6 mRNA and SA-β-gal activity were suppressed,\nbut not fully abolished, by miRNA inhibitors or NF-κB p65 knockdown without affecting REDD1\nprotein levels (Fig. 3f–h; Supplementary Figs. 3f, 4j). These data indicate that IL-6 expression\noccurs via two complementary mechanisms: (i) direct REDD1–NF-κB activation (Lines 330–\n340) and (ii) the miRNA–eNOS/SIRT1–senescence axis, forming a feed-forward loop that\namplifies the expression of SASP genes, including IL-6 (Lines 454–458). Fig. 10g was updated,\nand Fig. 3m removed accordingly.\n(3) REDD1 can also promote senescence independently of miRNAs by regulating NF-κB-\nresponsive senescence genes, including Apod, Gadd45, Bcl2, and Prkcd (Ref. 25, Lines 418–\n421).\n(4) Additionally, REDD1 may enhance IL-6 expression through NF-κB-independent pathways,\nas evidenced by minimal IL6 mRNA induction in ECs overexpressing the NF-κB-inactive\nREDD1KKAA mutant (Fig. 3m). This statement has been incorporated into the Discussion\nsection (Lines 379–384).\n(5) REDD1 protein levels are now included in Fig. 3f and 3k, with quantification in\nSupplementary Figs. 4k and 6b, confirming that REDD1 expression was unaffected by miRNA\ninhibition.\nComment #4: Positive controls are missing for the REDD1KKAA mutant protein in Fig. 3, 4, and\n6. While it is clear that the mutant does not activate EC senescence, it should be demonstrated\nthat it still acts to suppress other NF-κB-independent downstream effectors of REDD1 (e.g.\nmTORC1). One possibility is that the mutant protein is simply misfolded. These controls would\nalso help to ground the speculative statements included in the Discussion (lines 324-332).\nResponse: (1) We thank the Reviewer for raising this important point regarding positive\n3\ncontrols for the REDD1KKAA mutant protein. In our previous studies, we confirmed that\nREDD1KKAA fails to activate atypical NF-κB.\n(2) While REDD1 is known to inhibit mTORC1, it also regulates various cellular functions\nthrough interactions with multiple proteins (Ref. 18). Previous studies have shown that genetic\nand pharmacological mTORC1 inhibition can ameliorate cellular senescence, promote\nlongevity, and improve vascular function (Refs. 28–33). However, in this study, REDD1\noverexpression unexpectedly induced vascular senescence and dysfunction, indicating that\nthese effects are independent of mTORC1 signaling (Discussion; Lines 358–369).\n(3) In addition, REDD1 has been reported to induce oxidative stress and endothelial\nsenescence via interaction with TXNIP (Refs. 18, 22). To provide a positive control for\nREDD1KKAA function, we assessed its ability to bind TXNIP and confirmed that its binding ability\nwas comparable to that of wild-type REDD1 (Supplementary Fig. 6a; Lines 170–174),\ndemonstrating that the mutant protein is properly folded and retains functional competence in\nNF-κB-independent pathways. This indicates that the protective effects of Redd1KKAA are due\nto its inability to activate atypical NF-κB signaling and are independent of mTORC1 inhibition\nor protein misfolding, and this clarification has been incorporated into the Discussion section\n(Lines 364–369).\n(4) We also discussed the possible NF-κB-independent mechanisms of REDD1, including\nTXNIP-mediated oxidative stress, VEGFR-2/3 downregulation, and GRP75-mediated\nmitochondrial ROS generation, in the Discussion section (Lines 376–391).\nComment #5: The manuscript provides no evidence for a causal role of eNOS or SIRT in diet-\ninduced hypertension in mice. The only data that goes beyond association is found in Fig. 2j,\nwhich supports its role in REDD1-mediated EC senescence and Supplemental Fig. 5. Authors\nshould be careful not to overstate the findings related to eNOS in Supplemental Fig. 5, as the\nstudy was performed with healthy mice. Support for a causal role of SIRT1 in REDD1-\nmediated EC senescence and hypertension is lacking. Line 190-191: The speculatory clause\nregarding the potential role of SIRT1 is unnecessary. The title of Supplementary Fig. 3 also\noverstates the causal role, as only an association is demonstrated in the data. Also see line\n199.\nResponse: (1) We thank the Reviewer for raising the important point regarding the lack of\ndirect evidence for a causal role of eNOS or SIRT1 in REDD1-mediated vascular senescence\nand diet-induced hypertension in mice. We have revised the manuscript to avoid\noverstatement of the causality and clearly report the observed associations.\n(2) Although direct in vivo causal evidence is lacking, Fig. 2j shows that REDD1-induced EC\n4\nsenescence is significantly attenuated by SIRT1 overexpression or exogenous NO,\nsuggesting that downregulation of SIRT1 and the eNOS–NO axis is associated with REDD1-\nmediated pro-senescent effects. Previous studies also support well-characterized roles of\nSIRT1 and eNOS in endothelial senescence and vascular homeostasis (Refs. 12, 16, 17, 24,\n39). Relevant background information has been incorporated into the Introduction and Results\nsections (Lines 66–69, 76–80, 330–340)\n(3) Speculative language has been removed as follows (Lines 201–205): “Compared with\nWT mice treated with Ad-control, eNOS–/– mice receiving Ad-control exhibited higher aortic\nSA-β-gal activity and p53 expression, without changes in SIRT1 levels. Upon Ad-REDD1\ndelivery in eNOS⁻/⁻ mice, SIRT1 levels were reduced and p53 expression was markedly\nenhanced (Supplementary Fig. 8d, e).”\n(4) Original Line 199 revised as follows (Lines 211–214): “These results suggest that\nREDD1 overexpression induces vascular senescence and hypertension in vivo, accompanied\nby increased miR-214-3p levels and decreased eNOS and SIRT1 expression.”\n(5) Supplementary Fig. 3 title revised: “REDD1-induced EC senescence is modulated by\nNF-κB p65 knockdown, exogenous NO, and SIRT1 overexpression.”\n(6) These revisions clarify that REDD1 modulates EC senescence partly via SIRT1 and eNOS,\nbut the current data mainly show associations and modulatory effects, not definitive in vivo\ncausality. Future studies employing SIRT1–/– and eNOS–/–/SIRT1–/– mice will be necessary to\ndirectly establish their causal roles in REDD1-mediated vascular senescence and\nhypertension under obese conditions.\nComment #6: Figure 5: Care should be taken with the text to be sure that the data are not\noverstated. Dependence of REDD1 deficiency on eNOS and SIRT1 in this figure is not\ndemonstrated as suggested by figure’s title. Also, REDD1 deletion looks to partially restore\nchanges seen in the DIO model. It is not clear that statistical analysis was performed between\nlean and DIO-Red1–/– groups, many of which appear to be significantly different. This likely\nsupports that REDD1 is a key driver of EC senescence and hypertension, but not entirely\nresponsible for the changes. Statistically significant differences between lean and DIO\ntransgenic mice should also be indicated in other figures.\nResponse: (1) We thank the reviewer for highlighting the need to avoid overstatement of the\nrole of REDD1 in Figure 5. In response, the Results section, figure legend, and associated\ntext have been revised as follows:\n(2) The title and legend of Figure 5 (Line 5) have been revised to: “Redd1 loss attenuates\nDIO-induced vascular senescence and hypertension” and “Redd1 deficiency reduces EC\n5\nsenescence and hypertension in DIO mice, accompanied by partial reduction of miR-214-3p\nlevels and partial preservation of eNOS and SIRT1 expression”, respectively.\n(3) Statistical comparisons between lean and DIO-Redd1–/– groups have been performed for\nall measured parameters in Figure 5, with significant differences indicated. Similar statistical\nanalyses have been incorporated in all other relevant figures to ensure clarity and\ntransparency.\n(4) The conclusion statement in the Results section has been revised (Lines 235–238):\n“These results indicate that REDD1 deficiency is associated with attenuated obesity-induced\nEC senescence and hypertension, with partial reduction of miR-214-3p levels and partial\nrestoration of eNOS and SIRT1 expression, suggesting that REDD1 contributes to, but is not\nsolely responsible for, these phenotypes.”\n(5) These revisions clarify that REDD1 is an important contributor to obesity-induced vascular\nsenescence and hypertension, while other factors also play a role, avoiding overstatement of\nits effects.\nComment #7: In this manuscript the authors show that miR-214-3p loss alleviated DIO-\ninduced vascular dysfunction. Based on prior literature this might not be an endothelial-specific\neffect. miRNA-214-3p in the kidney contributes to the development of hypertension (PMCID:\nPMC6171279). Recent studies have also shown that upregulated miRNA-214 also modulates\nthe senescence of vascular smooth muscle cells in carotid artery stenosis (PMCID:\nPMC7227274). Importantly, miRNAs often have a dual regulatory role. Studies have\npreviously demonstrated that miRNA-214-3p protects against myocardial ischemia-\nreperfusion injury (PMCID: PMC10066509) and knockdown of microRNA-214-3p promotes\ntumor growth and Epithelial-Mesenchymal Transition in prostate cancer (PMCID:\nPMC8656576). These are important considerations that should must be addressed\nexperimentally.\nResponse: (1) We thank the Reviewer for highlighting that miR-214-3p may exert effects\nbeyond ECs and display dual, context-dependent regulatory roles. miR-214-3p likely has both\ndirect effects in ECs and indirect effects via other vascular or non-vascular cell types, as it is\npredicted to target over 100 genes (TargetScan analysis).\n(2) As suggested, we added the following to the Discussion section (Lines 427–432):\n“miR-214-3p exhibits pleiotropic, context-dependent effects beyond the endothelium,\nregulating various genes, such as Quaking, Kdm3a, Cdh1, and Ptk6, and contributing to renal\nhypertension, vascular smooth muscle cell senescence, myocardial protection, and cancer\ngrowth inhibition50–52. Although cell type-specific functions were not examined, both miRNAs\n6\nlikely affect EC dysfunction primarily through downregulation of the EC-specific eNOS–NO\naxis and ubiquitously expressed SIRT1.”\n(3) We further expanded the Discussion to acknowledge alternative or additional\ndownstream pathways (Lines 433–440): “However, additional targets and pathways cannot\nbe excluded. For instance, miR-155-5p may regulate the senescence-associated TERF1–\np21/p16 axis53, while miR-214-3p may influence quaking-dependent vascular pathways,\nincluding the downregulation of TERT/TERF1–p21/p16 and VEGFA/bFGF50,54. Notably, RNA-\nseq analysis of miR-214-3p-knockdown cells revealed upregulation of numerous genes\nassociated with angiogenesis, cell proliferation, and cell migration52. Collectively, these\nfindings suggest that miR-214-3p impairs endothelial function not only through the\nsuppression of eNOS and SIRT1 but also via additional target genes that regulate vascular\nhomeostasis.”\n(4) These revisions ensure that the manuscript clearly acknowledges both the endothelial-\nspecific and broader systemic roles of miR-214-3p, while emphasizing that the current study\nprimarily focused on endothelial mechanisms, with contributions from other cell types remain\npossible.\nComment #8: A previous study has also demonstrated a role for REDD1 in doxorubicin-\nelicited endothelial cell dysfunction through downregulation of VEGFR-2/3 expression (PMCID:\nPMC8568908). Inhibition of VEGF can induce cellular senescence (PMID: 21618508). Can\nthe authors speculate on this contrary observation?\nResponse: (1) We thank the reviewer for highlighting the previous study that showed that\nREDD1 mediates doxorubicin-induced endothelial dysfunction through VEGFR-2/3\ndownregulation [Refs. 34 (PMCID: PMC8568908), 35]. The VEGF/VEGFR axis is a central\nregulator of vascular homeostasis, and VEGF inhibition (e.g., by Bevacizumab) is well known\nto induce endothelial senescence and hypertension in both experimental models and patients\n[Refs. 36 (PMID: 21618508), 37].\n(2) In this study, we identified the REDD1–NF-κB–miR-155-5p/miR-214-3p–eNOS/SIRT1 axis\nas the predominant mechanism driving obesity-associated endothelial dysfunction and\nsenescence. We have added the following statement in the Discussion section (Lines 376–\n384); “In cellular senescence, REDD1 can promote cellular dysfunction, including endothelial\nsenescence, independently of NF-κB, through mechanisms including TXNIP-mediated\noxidative stress, VEGFR-2/3 downregulation, and GRP75-mediated mitochondrial ROS\ngeneration4,18,22,23,34–37. These NF-κB-independent pathways may contribute to EC\nsenescence and hypertension under various pathological stress conditions36,37, although their\nroles in metabolic contexts remain unclear. Our data show that the atypical NF-κB-inactive\nbinding, indicating that NF-κB-independent pathways contribute modestly under metabolic\nstress.”\n(3) These revisions clarify the potential involvement of NF-κB-independent pathways in\nREDD1-mediated EC senescence and vascular dysfunction, emphasizing that their\ncontribution is modest under metabolic stress.\nComment #9: A previous study has reported that Kallistatin reduces vascular senescence and\naging by regulating microRNA‐34a‐SIRT1 pathway (PMID: 29992759). Another study has\nshown that reduced expression of microRNA-130a promotes endothelial cell senescence\n(PMCID: PMC7346016). Since microRNA‐34a is upregulated (PMID: 22988100) and\nmicroRNA-130a is reduced (PMID: 23396142) in obese patients, the authors should try to\nrule out the role of other miRNAs in obesity-induced vascular senescence. Importantly, clinical\nevidence for increased miR-155-5p in obese patients would greatly enhance the impact of this\nstudy.\nResponse: (1) We thank the reviewer for noting the potential roles of other miRNAs, including\nmiR-34a and miR-130a, in obesity-induced vascular senescence. Although miR-31-5p, miR-\n34a-5p, and miR-130a-3p can modulate NF-κB, eNOS, and SIRT (Refs. 5, 10, 55–57), their\nlevels were only partially, and not significantly, increased in aortic tissue of DIO mice\n(Supplementary Fig. 13), suggesting that these miRNAs are unlikely to play a significant role\nin obesity-induced vascular dysfunction under DIO. This has been incorporated into the\nDiscussion (Lines 444–449): “Because individual miRNAs can target multiple genes and\nsingle genes can be regulated by several miRNAs, other NF-κB- or obesity-related miRNAs,\nsuch as miR-31-5p, miR-34a-5p, miR-130a-3p, may also contribute to vascular dysfunction\nby modulating NF-κB, eNOS, and SIRT15,10,55–57. However, their levels were only partially, but\nnot significantly, increased in aortic tissue of DIO mice (Supplementary Fig. 13), suggesting\nthat these miRNAs may not play a major role in obesity-induced vascular dysfunction.”\n(2) We have added the following statement regarding the clinical relevance of REDD1, miR-\n155-5p, miR-214-3p, eNOS, and SIRT1 in patients with obesity, hypertension, and metabolic\ndisease (Lines 449–454): “Consistent with our findings, clinical studies show that REDD1,\nmiR-155-5p, and miR-214-3p are elevated in patients with obesity, hypertension, or metabolic\ndisease38,46,48,49,57–59,60–62, whereas eNOS and SIRT1 are consistently reduced43,60–62. These\nfindings support the translational relevance of the REDD1–atypical NF-κB–miRNA axis,\nsuggesting that similar mechanisms operate in human obesity-associated vascular disease.”\n(3) These revisions clarify that while other miRNAs could theoretically contribute to vascular\ndysfunction, our data indicate that miR-155-5p and miR-214-3p are the primary mediators in\nvascular disease.\nComment #10: Figure 6: It is unclear if levels of obesity-related factors are similar in DIO WT\nversus REDD1KKAA mice.\nResponse: (1) We thank the reviewer for raising this important point regarding obesity-related\nfactors in different mouse models. To address this, we analyzed circulating obesity-related\nfactors under DIO conditions in three models: WT versus Redd1KKAA, Redd1fl/fl versus\nRedd1ΔEC, and WT versus miR-214-3p–/–. Consistent with a prior study (Ref. 20), Redd1KKAA\nmice exhibited significantly lower levels of these factors compared with WT mice, whereas no\nsignificant differences were observed between Redd1fl/fl versus Redd1ΔEC mice or WT versus\nmiR-214-3p–/– mice.\n(2) These findings suggest that endothelial REDD1 is a key mediator of vascular senescence\nand hypertension under obese conditions, supporting its potential as a therapeutic target.\nThe new data are presented in Supplementary Fig. 10a–c, and corresponding statements have\nbeen incorporated into the Results section (Lines 260–263, 275–277, 299–300).\nComment #11: The rigor of western blot analysis is poor with often only single replicates\nshown and no quantifications.\nResponse: We thank the reviewer for highlighting the need to improve the rigor of our western\nblot analyses. To address this, all experiments were independently repeated at least three\ntimes, protein band intensities were analyzed statistically using ImageJ, and the resulting data\nare now presented in the corresponding Supplementary Figures, enhancing the reproducibility\nand reliability of our findings and ensuring that the conclusions drawn from the western blot\nexperiments are robust.\nComment #12: While deletion of REDD1 from birth is a useful way to delineate pathways\nbeing regulated, it is not a therapeutically translational model as the disease is induced later\nin life. For the model to be therapeutically translational, the authors might try to suppress\nREDD1 after the mice are made obese and DIO symptoms such as elevated serum parameters are\nobserved.\nResponse: We thank the reviewer for this insightful comment. We agree that constitutive\nendothelial REDD1 deletion from birth may not fully reflect a therapeutic context. To address\nthis, we generated post-obesity EC-specific knockout mice (DIO-Redd1ΔEC-post) using VE-\ncadherin-CreERT2:Redd1fl/fl. Diet-induced obesity was first established by 14 weeks of high-\n9\nfat diet and confirmed by elevated serum leptin and triglyceride levels (Supplementary Fig.\n11a, b). Tamoxifen was then administered to induce endothelial REDD1 deletion. Two weeks\nafter tamoxifen, vascular senescence, ACh-induced mesenteric arterial vasorelaxation,\nsystolic blood pressure, and related molecular markers were significantly improved compared\nwith DIO-Redd1fl/fl controls (Supplementary Fig. 11c–i), closely resembling the protective\neffects observed in constitutive DIO-Redd1ΔEC mice. These data have been added to the\nResults section (Lines 287–295) to highlight the therapeutic relevance of targeting endothelial\nREDD1. Collectively, our findings indicate that REDD1 is a key mediator of DIO-induced\nvascular senescence and hypertension, independent of systemic metabolic alterations.\nMinor comments:\nComment #1: SIRT1 is misspelled in the title.\nResponse: We thank the reviewer for identifying this typographical error. The title has been\ncorrected to accurately reflect the proper spelling of SIRT1. We appreciate the reviewer’s\ncareful attention to detail.\nComment #2: In the methods section the authors state the miR-214-5p–/– mice were\ngenerated (line 484). All the data shown in the manuscript indicate that miR-214-3p–/– mice\nwere used. This potentially calls into question all studies reported in the miR-214-3p–/– mice,\nbut is hopefully just another typo.\nResponse: We thank the reviewer for highlighting this issue. The mention of miR-214-5p in\nthe Methods section (Line 521) was a typographical error. All experiments were conducted\nusing miR-214-3p–/– mice, and all data in the manuscript accurately reflect results from this\nmodel. The Methods section has been corrected accordingly, and we apologize for this\noversight while appreciating the reviewer’s careful reading.\nComment #3: Many gene names in Supplementary Table 2 are incorrectly formatted. These\ninclude hyphens and a random use of capitalization. REDD1 gene as Redd1, rather than\nDDIT4 is also unconventional.\nResponse: We thank the reviewer for highlighting issues with gene nomenclature in\nSupplementary Table 2. All gene names have been reviewed and corrected according to\ninternational standards, with human and mouse genes formatted per HGNC and MGI\nguidelines, respectively. Hyphens and capitalization have been standardized. The gene\nsymbol REDD1 has been retained throughout the manuscript because it is more widely used\n10\nin the literature than the official symbol DDIT4, particularly in studies of vascular and metabolic\nstress. Retaining REDD1 ensures consistency with results of prior research and facilitates\nreader comprehension. We appreciate the reviewer’s careful attention, which has improved\nthe accuracy and clarity of the manuscript.\nComment #4: Line 130: suggest editing “effectively rescued REDD1-mediated promoter\nactivity…”. The siRNA for p65 does not appear to rescue REDD1-dependent promoter activity,\nbut rather is preventing it.\nResponse: We thank the reviewer for this valuable clarification. Upon re-examining our data,\nwe confirm that siRNA targeting NF-κB p65 prevents, rather than rescues, REDD1-mediated\npromoter activity. Accordingly, the Results section has been revised (Lines 140–144). The\nupdated sentence now reads: “Notably, knockdown of NF-κB p65, but not IKK, inhibited\nREDD1-mediated NF-κB promoter activity (Supplementary Fig. 3b, c) and attenuated REDD1-\ninduced changes in eNOS, SIRT1, p53/p21, SA-β-gal activity, and IL-6 mRNA levels, although\nresidual SA-β-gal activity and IL-6 mRNA levels remained significantly elevated (Fig. 2h, i and\nSupplementary Fig. 3d–f).” We appreciate the reviewer’s constructive comment, which has\nhelped improve the clarity and accuracy of the Results section.\nComment #5: Sup. Fig. 2c: suggest editing the confusing location of “ns” and bar between\ngroups 2 and 4.\nResponse: We thank the reviewer for this helpful suggestion. To address the concern, we\nreconfirmed the statistical significance for all relevant groups and revised Supplementary Fig.\n3c, adjusting the “ns” labels and the positions of the bars to clearly indicate all comparisons,\nincluding between groups 2 and 4. This revision ensures that the figure is clear and resolves\nthe previous source of confusion.\nComment #6: Line 143: The REDD1-dependent increase in SA-β-gal was reduced but not\n“prevented”.\nResponse: We thank the reviewer for this valuable suggestion. In response, we have revised\nthe description in the Results section to more accurately reflect our data. The revised text\n(Lines 151–157) now reads: “Knockdown of DICER, a key enzyme for miRNA biosynthesis,\nsubstantially reversed the REDD1-induced suppression of eNOS, SIRT1, and NO production,\nand the increase in intracellular NO was abolished by L-NAME treatment (Fig. 3a, b and\nSupplementary Fig. 4a, b). DICER knockdown also attenuated REDD1-mediated increases in\nSA-β-gal activity, miR-155-5p and miR-214-3p levels, and IL6 mRNA levels; however, SA-β-\n11\ngal activity and IL6 mRNA levels remained partially but significantly elevated (Fig. 3c–e and\nSupplementary Fig. 4c).” This revision more accurately reflects the extent of the effects of\nDICER knockdown on REDD1-mediated changes.\nComment #7: Line 145: incorrect description. Potentially missing phrase “which was\nprevented by”.…knockdown of p65.\nResponse: We thank the reviewer for pointing out this issue. We agree that the original\ndescription was inaccurate. In response, we re-examined the data (and performed additional\nconfirmatory experiments) and revised the Results section to accurately describe the effects\nof NF-κB p65 knockdown on REDD1-mediated changes. The revised text (Lines 157–160)\nnow reads: “As both miRNAs are regulated by NF-κB activation9,11, REDD1-mediated\nincreases in miR-155-5p and miR-214-3p levels were suppressed by knockdown of NF-κB\np65, but not IKKβ (Supplementary Fig. 4d, e), suggesting that REDD1 promotes miR-155-5p\nand miR-214-3p biogenesis via atypical NF-κB activation.” This revision clarifies that p65,\nbut not IKKβ, is required for REDD1-mediated induction of miR-155-5p and miR-214-3p.\nComment #8: Line 290: “Despite being more readily detected at its mRNA level…” This\nstatement is unclear. Are the authors implying a lack of coordination between mRNA and\nprotein?\nResponse: We thank the reviewer for pointing this out. To avoid potential confusion regarding\nthe relationship between mRNA and protein levels, we have removed the original sentence\n(“Despite being more readily detected at its mRNA level…”) from the Discussion. This revision\nensures that the text remains clear and focusses on the main message.\nComment #9: The absence of cardiovascular deficits in diabetic Redd1–/– mice fed a high-fat\ndiet has already been demonstrated and linked to NF-κB signaling (PMID: 38928166)\nResponse: (1) We thank the reviewer for highlighting this important point. Indeed, previous\nstudies demonstrated that Redd1 deletion protects against cardiac functional deficits in\nstreptozotocin-induced diabetic mice, with effects linked to NF-κB signaling (PMID: 38928166,\ncited as Ref. 27). Similarly, in this study, REDD1-induced EC senescence in HAECs was\nsubstantially reversed by NF-κB p65 knockdown, whereas IKKβ knockdown had little effect.\nThese findings suggest that, in ECs under metabolic stress, REDD1 primarily engages an\natypical, IKK-independent NF-κB pathway, which may differ from the GSK3β-mediated\ncanonical NF-κB activation reported in cardiomyocytes and cardiac tissues of streptozotocin-\ninduced diabetic mice.\n12\n(2) Accordingly, the following sentences were incorporated into the Discussion section (Lines\n344–357): “Consistently, NF-κB p65 knockdown substantially reversed REDD1-induced EC\nsenescence in HAECs, whereas IKKβ knockdown had little effect. Pharmacological inhibition\nof IKKβ partially ameliorated vascular senescence and dysfunction in both DIO-WT and DIO-\nRedd1–/– mice, showing greater efficacy in DIO-WT mice. Notably, TNF-, a canonical NF-κB\nactivator, induced EC senescence independently of REDD1, likely via canonical NF-κB-\ndependent induction of miR-155-5p and miR-214-3p. These results suggest that metabolic\nstress-induced REDD1 engages an atypical NF-κB pathway, which appears mechanistically\ndistinct from the GSK3β-mediated canonical NF-κB mechanism reported in cardiomyocytes\nof streptozotocin-induced diabetic mice27. Our results further imply that both the REDD1-\nmediated atypical NF-κB axis and the canonical NF-κB pathway are activated under obese\nconditions. However, the atypical pathway may be preferentially triggered and facilitate\ncanonical NF-κB activation via direct or indirect cytokine signaling, indicating a functional\ninterplay between the two pathways. Further studies using EC-specific Ikkβ–/– mice are\nwarranted to delineate the precise role of the canonical pathway.”.\n(3) These revisions emphasize the cell type- and context-specific roles of REDD1–NF-κB\nsignaling in metabolic stress-induced cardiovascular dysfunction, clarifying the distinction\nbetween atypical and canonical NF-κB activation.\nIn this manuscript, the authors investigated the function of REDD1 in regulating obesity-\ninduced endothelia cell (EC) senescence and hypertension. They found that HFD feeding\nincreased REDD1 expression in aortic endothelium in mice and obesity promote EC\nsenescence in a REDD1-dependent manner. They further revealed that REDD1 plays a\ncentral role in the pathogenesis of obesity-induced EC senescence and hypertension by\nstimulating NF-κB-dependent miR-155-5p/214-3p expression, which leads to the\ndownregulation of eNOS and SIRT1 expression. The manuscript included comprehensive in\nvitro and in vivo data. However, there are several issues with the mouse models the authors\nused and the described underlying mechanisms. I have the following specific comments:\nComment #1: The authors showed that systemic delivery of Ad-REDD1 but not Ad-REDD1KKAA\ninduces EC senescence and hypertension. It is not clear whether Ad-REDD1 and Ad-REDD1KKAA\ncan efficiently target other tissues in addition of aorta. What is the tissue distribution pattern of\nRedd1 or REDD1KKAA after delivery? REDD1overexpression has been shown to stimulate\npreadipocyte differentiation and proinflammatory cytokine expression through atypical IKK-independent\nNF-κB activation in adipose tissue (Nat Commun. 13, 6303 (2022)). If Ad-REDD1 and\nAd-REDDKKAA delivery also led to increased RREDD1 or REDD1KKAA expression in adipose\ntissue, this may affect the obesity and its’s associated phenotypes. The authors need to\ncharacterize the obesity and metabolic phenotypes carefully in those mice.\nResponse: (1) We thank the reviewer for this insightful comment. To address the concerns\nregarding tissue distribution and potential metabolic effects, we analyzed REDD1 expression\nacross multiple tissues and measured circulating obesity-related factors in mice injected with\nAd-REDD1 or Ad-REDD1KKAA. The new data are presented in Supplementary Fig. 8a/b and\ndescribed in the revised Results section (Lines 186–192) as follows: “To investigate the in vivo\nrole of REDD1 in vascular dysfunction, male mice were intravenously injected with Ad-REDD1\nor Ad-REDD1KKAA. In both groups, Redd1 mRNA levels were comparably upregulated across\nmost tissues, showing the highest levels in the liver, markedly increased expression in the\nheart and aorta, and minimal expression in adipose tissue (Supplementary Fig. 8a). Serum\nlevels of obesity-related factors, such as leptin, resistin, and triglycerides, showed no\nsignificant changes, whereas TNF- was elevated only in Ad-REDD1-injected mice\n(Supplementary Fig. 8b).”\n(2) These findings indicate that adenoviral delivery did not induce major obesity-related or\nmetabolic changes, minimizing the potential confounding effects from adipose tissue. Instead,\n14\nthe results support a specific role of REDD1 in promoting endothelial senescence and\nhypertension.\nComment #2: In Fig. 5, the authors showed Redd1 deficiency prevents DIO-induced EC\nsenescence and hypertension. Again, did Redd1 deficiency affect the DIO directly? Obesity\nand metabolic phenotypes of those mice under chow and HFD feeding condition need to be\ncharacterized.\nResponse: (1) We thank the reviewer for this important comment. Because it has been\npreviously reported that Redd1–/– mice exhibited protection against obesity and metabolic\nphenotypes compared with WT mice under HFD feeding (Ref. 20), we further analyzed\ncirculating obesity-related factors under lean and DIO conditions in three mouse models: WT\nversus Redd1KKAA, Redd1fl/fl versus Redd1ΔEC, and WT versus miR-214-3p–/–. Consistent with\na previous study in DIO-Redd1–/– mice (Ref. 20), obesity-related factors were significantly\nreduced in Redd1KKAA mice compared with WT controls under DIO conditions. In contrast, no\nsignificant differences were observed between Redd1fl/fl versus Redd1ΔEC mice or between WT\nand miR-214-3p–/– mice. Importantly, these parameters showed no significant differences\nbetween genotypes under lean conditions.\n(2) These results suggest that the vascular protective effects observed in Redd1ΔEC and miR-\n214-3p–/– mice are not due to changes in obesity or metabolic phenotypes, but rather reflect\nthe direct role of endothelial REDD1 in promoting vascular senescence and hypertension.\n(3) The new data are presented in Supplementary Fig. 10a–c, and the Results section has\nbeen updated accordingly (Lines 260–263, 275–277, 299–300).\nComment #3: The authors generated EC-specific Redd1 mouse model which could address\nsome of above concerns. However, the manuscript did not include basic characterization of\nthese mice. Did deficiency of Redd1 in ECs protect mice from DIO, systemic inflammation,\nand other metabolic disorder. It is plausible that those factors can contribute indirectly to the\nobserved EC senescence and hypertension phenotypes.\nResponse: (1) We thank the reviewer for this important point. A prior study reported that\nRedd1–/– mice exhibited protection against meta-inflammation compared with WT mice under\nDIO conditions (Ref. 20). As described in the response to Comment #2, we also analyzed\nserum TNF- levels under lean and DIO conditions in three mouse models: WT versus\nRedd1KKAA, Redd1fl/fl versus Redd1ΔEC, and WT versus miR-214-3p–/–. DIO increased serum\nTNF- levels in WT mice, which was prevented in Redd1KKAA mice, but not in Redd1ΔEC or\nmiR-214-3p–/– mice; under lean conditions, no significant differences were observed between\n15\nany of the genotypes and WT mice.\n(2) These results indicate that the vascular protective effects observed in Redd1ΔEC and miR-\n214-3p–/– mice occur independently of systemic inflammatory changes.\n(3) The new data are presented in Supplementary Fig. 10a–c, and the Results section has\nbeen updated accordingly (Lines 260–263, 275–277, 299–300).\nComment #4: miR-155-5p and miR-214-3p have been well known to play diverse roles in\nregulating various gene expression and are also linked to cancer, inflammation, and many\nphysiological processes. The authors should explore how the changed miR-155-5p and miR-\n214-3p affect the different gene/pathways in addition to eNOS and SIRT1. For example,\nexperiments would give a better overview of the impact of miR-155-5p and miR-214-3p on EC\ngene expression. Do other genes/pathways contribute to observed EC senescence or\ndysfunction?\nResponse: (1) We thank the reviewer for highlighting the broader roles of miR-155-5p and\nmiR-214-3p beyond SIRT1 and eNOS. Although we did not perform RNA-seq in this study,\nprevious studies, including RNA-seq data, have shown that these miRNAs regulate multiple\ngenes and pathways that influence endothelial senescence and vascular dysfunction.\n(2) Regarding additional targets and pathways of miR-155-5p and miR-214-3p beyond SIRT1\nand eNOS, we have added the following statement to the Discussion section (Lines 433–440):\n“However, additional targets and pathways cannot be excluded. For instance, miR-155-5p\nmay regulate the senescence-associated TERF1–p21/p16 axis53, while miR-214-3p may\ninfluence quaking-dependent vascular pathways, including the downregulation of\nTERT/TERF1–p21/p16 and VEGFA/bFGF50,54. Notably, RNA-seq analysis of miR-214-3p-\nknockdown cells revealed upregulation of numerous genes associated with angiogenesis, cell\nproliferation, and cell migration52. Collectively, these findings suggest that miR-214-3p impairs\nendothelial function not only through the suppression of eNOS and SIRT1 but also via\nadditional target genes that regulate vascular homeostasis.”\n(3) We also incorporated the following information into the limitations section of Discussion\nsection (Lines 478–479): “Finally, additional targets of miR-155-5p and miR-214-3p should be\nidentified using RIP-seq or CLIP-seq approaches that exploit target site complementarity.”\nIn this study, authors analyzed the roles of REDD1 in mediating endothelial dysfunction due\nto obesity, and found that REDD1 promotes NF-κB signaling, which alters eNOS and SIRT1\nvia miR-214-3p. This mechanism is lengthy and possibly REDD1 can directly exert effects on\nendothelial cells considering that REDD1 is a stress protein known to induce senescence.\nThere are several major problems:\nComment #1: The evidence supporting the involvement of NF-κB signaling is weak. Also, NF-\nκB signaling is a generic inflammatory pathway which can induce many changes, not only\nmiR-214-3p and others.\nResponse: (1) We thank the reviewer for this important comment. Additional experiments\nshowed that obesity-related factors, palmitic acid and oxLDL increased miR-155-5p and miR-\n214-3p levels in HAECs and MAECs. This induction was effectively inhibited by NF-κB p65\nknockdown and substantially reduced by REDD1 or IKKβ silencing, although residual miRNA\nlevels remained significant (Supplementary Fig. 7; Lines 176–183). These results indicate that\nREDD1-dependent atypical NF-κB activation serves as an important upstream driver of\nmiRNA biogenesis, although IKK-dependent NF-κB signaling also contributes.\n(2) Moreover, an additional experiment using the IKK inhibitor Bay 11-7082 showed that\ncanonical NF-κB signaling is also activated but less critical under DIO conditions (Fig. 6a–h;\nSupplementary Fig. 9a, b; Lines 240–256). The interplay between atypical and canonical NF-\nκB pathways may further exacerbate EC senescence and vascular dysfunction by enhancing\nmiRNA biogenesis and cytokine expression (Fig. 10g; Lines 330–340).\n(3) Supporting evidence includes several key observations: NF-κB p65 knockdown, but not\nIKKβ silencing, attenuated REDD1-mediated NF-κB activation and miRNA expression,\neNOS/SIRT1 downregulation, and EC senescence (Fig. 2h, i; Supplementary Fig. 4d, e);\noverexpression of REDD1, but not the NF-κB-inactive mutant REDD1KKAA, increased miRNA\nlevels and EC senescence (Fig. 3j–m); and DIO-induced vascular dysfunction occurred in WT\nmice but was significantly reduced in Redd1KKAA mice (Fig. 7).\n(4) NF-κB is a key driver of cellular senescence (Refs. 2, 29, 31) and may regulate multiple\ndownstream targets beyond miR-155-5p/miR-214-3p, including NF-κB-responsive or obesity-\nassociated miR-31-5p, miR-34a-5p, and miR-130a-3p, which may influence EC function via\nNF-κB, eNOS, and SIRT1 (Refs. 5, 10, 55–57). However, our qRT-PCR analysis additionally\nreveal that these miRNAs were not significantly elevated in the aortic tissue of DIO-WT mice\n(Supplementary Fig. 13; Lines 444–449), suggesting that they may not be critically involved\nin REDD1-dependent or obesity-mediated EC senescence and vascular dysfunction.\n17\n(5) REDD1-induced NF-κB activation itself can induce EC senescence independently of\nmiRNAs. Although not directly examined, we discuss this possibility in the revised manuscript\nas follows (Lines 418–421): “REDD1 can also promote senescence by regulating NF-κB-\nresponsive senescence genes, including Apod, Gadd45, Bcl2, and Prkcd25, independently of\nmiR-155-5p and miR-214-3p; this suggests that REDD1 induces vascular dysfunction via\natypical NF-κB-dependent mechanisms.”\nComment #2: REDD1 can induce cell senescence directly with mechanisms not only involving\nNF-κB signaling and eNOS.\nResponse: (1) We thank the reviewer for highlighting that REDD1 may induce EC senescence\nthrough mechanisms beyond NF-κB and eNOS/SIRT1. Although we did not directly\ninvestigate alternative pathways in this study, several mechanisms have been proposed in\nprevious work.\n(2) We agree that REDD1 can induce vascular dysfunction independently of NF-κB, albeit\nmodestly. As described in the Discussion section (Lines 358–369, 376–384), this may involve\nmechanisms, such as NF-κB–independent mTOR inhibition, TXNIP-mediated oxidative stress,\nVEGFR-2/3 downregulation, and mitochondrial dysfunction, which are directly regulated by\nREDD1. Our data suggest that mTOR inhibition is unlikely to be involved. In contrast, TXNIP-\nmediated oxidative stress, VEGFR-2/3 downregulation, and mitochondrial dysfunction may be\nrelevant and considered as NF-κB-independent additional mechanisms.\n(3) Since NF-κB can upregulate several senescence-associated genes (Ref. 25), we have\nadded the following information into the Discussion section (Lines 418–421). “REDD1 can\nalso promote senescence by regulating NF-κB-responsive senescence genes, including Apod,\nGadd45, Bcl2, and Prkcd25, independently of miR-155-5p and miR-214-3p; this suggests that\nREDD1 induces vascular dysfunction via atypical NF-κB-dependent mechanisms.”\n(4) Taken together, these findings indicate that the REDD1–atypical NF-κB–eNOS/SIRT1 axis\nplays a central role in endothelial senescence, while NF-κB–independent mechanisms may\nalso contribute under specific vascular stress conditions, albeit to a lesser extent.\nComment #3: More evidence is needed in the involvement of eNOS and SIRT1.\nResponse: (1) We thank the reviewer for emphasizing the need for additional evidence on\nthe involvement of eNOS and SIRT1. The eNOS–NO axis and SIRT1 are well-established\nregulators of endothelial senescence and vascular homeostasis (Refs. 12, 15, 17, 24, 39–41,\n55). This information has now been incorporated into the Introduction, Results, and Discussion\nsections.\n18\n(2) In this study, REDD1 was shown to suppress the expression of eNOS and SIRT1 via NF-\nκB–dependent induction of miR-155-5p and miR-214-3p. REDD1 increased SA-β-gal activity\nand reduced protein expression and 3′-UTR activities of eNOS and SIRT1; these effects were\nreversed by DICER knockdown or miRNA inhibition, whereas miRNA mimics reproduced the\nsuppressive effect (Fig. 3a–c; Supplementary Figs. 4a–g and 5). These functional experiments\nfurther showed that REDD1-induced endothelial senescence was alleviated by an NO donor\n(DETA-NO) or SIRT1 overexpression, but not by a dominant-negative SIRT1, confirming the\nessential roles of eNOS and SIRT1 (Fig. 2j).\n(3) Furthermore, Ad-REDD1 delivery induced vascular senescence and hypertension in WT\nand iNOS–/– mice but had minimal effects in eNOS–/– mice, highlighting the critical role of eNOS\nin REDD1-mediated vascular dysfunction (Fig. 4; Supplementary Fig. 8d–i).\n(4) Taken together, these findings provide direct and functional evidence that REDD1\npromotes endothelial senescence and vascular dysfunction through NF-κB–mediated\nsuppression of eNOS and SIRT1, thereby directly addressing the reviewer’s concern.\nComment #4: It remains unclear why REDD1 is activated in endothelial cells due to obesity.\nIt might be possible that obesity-induced inflammation activates REDD1, which then induces\nendothelial senescence, instead of REDD1 inducing inflammation.\nResponse: (1) We thank the reviewer for raising this important point. Data from this study\nshow that REDD1 expression is elevated in the vascular endothelium of obese mice (Fig. 1a,\nb), coinciding with increased circulating metabolic and adipose tissue–derived hormonal\nfactors (Supplementary Fig. 1a). Exposure of ECs to these factors increased REDD1\nexpression, miR-155-5p/miR-214-3p levels, SA-β-gal activity, and p53/p21, all of which were\nattenuated by REDD1 knockdown (Fig. 1d–h; Supplementary Figs. 1e, 7). These results\nindicate that metabolic and hormonal changes, rather than inflammatory cytokines, drive\nREDD1 upregulation in ECs.\n(2) In contrast, obesity-associated inflammatory cytokines (TNF- and IL-6) did not increase\nREDD1 expression. TNF-α induced EC senescence without affecting REDD1, and its effect\nwas reversed by IKKβ knockdown but not by REDD1 knockdown (Supplementary Fig. 1f).\nSimilarly, IL-6 did not elevate REDD1 mRNA levels (Supplementary Fig. 1g).\n(3) Together, these findings suggest that REDD1 activation in obesity is primarily mediated by\ncirculating metabolic and hormonal factors rather than inflammatory cytokines, as summarized\nin Fig. 10g.\nComment #1: Line 3: SITR1 should be SIRT1.\nResponse: We thank the reviewer for pointing out this typo. We have corrected “SITR1” to\n‘SIRT1’ in the revised manuscript.\nComment #2: Line 111: Please also test TNF-, a direct activator of NF-ΚB signaling.\nResponse: We thank the reviewer for this suggestion. We tested TNF-, a direct activator of\nNF-κB signaling, in endothelial cells. The new data have been incorporated into\nSupplementary Fig. 1f, and the corresponding results have been added to the revised\nmanuscript (Lines 120–124); “Notably, TNF-, an obesity-induced inflammatory cytokine,\nincreased senescence markers without inducing REDD1 expression, and these effects were\nblocked by IKKβ knockdown, but not by REDD1 knockdown (Supplementary Fig. 1f).\nSimilarly, IL-6 failed to induce REDD1 mRNA expression (Supplementary Fig. 1g).”\nComment #3: Line 171: There is insufficient evidence to support NF-κB involvement.\nResponse: (1) We thank the reviewer for this comment. Previous dtudies (Refs. 19, 20)\nshowed that REDD1 atypically activates NF-κB via IB binding (Refs. 19, 20). This study\ndemonstrate that REDD1 overexpression results in increased NF-κB-promoter activity,\nelevated miR-155-5p/miR-214-3p expression, downregulated eNOS/SIRT1 levels, and\nincreased SA-β-gal activity, and all these effects were inhibited by knockdown of NF-κB p65,\nbut not IKKβ (Fig. 2h, i; Supplementary Figs. 3–4).\n(2) In addition, we newly found that HAECs and MAECs exposed to obesity-related factors,\npalmitic acid and oxLDL, increased NF-κB–responsive miR-155-5p and miR-214-3p levels,\nwhich were effectively inhibited by NF-κB p65 knockdown and substantially reduced by\nREDD1 or IKKβ silencing, although residual miRNA levels remained significant\n(Supplementary Fig. 7; Lines 176–183). These results indicate that REDD1, induced by\nobesity-related factors, promotes EC senescence via NF-κB–dependent biogenesis of both\nmiRNAs targeting eNOS and SIRT1.\n(3) Although not directly examined, several additional pathways have been described in the\nrevised manuscript to discuss the possible involvement of REDD1 in DIO-induced endothelial\nand vascular dysfunction; (i) TXNIP-mediated oxidative stress, VEGFR-2/3 downregulation,\nand mitochondrial dysfunction (Lines 376–384); (ii) NF-κB-responsive expression of\nsenescence-associated genes, such as Apod, Gadd45, Bcl2, and Prkcd (Lines 418–421);\nand\n20\n(iii) downregulation of quaking and its downstream signaling of TERT/TERF1–p21/p16 and\nVEGFA/bFGF (Lines 427–440).\n(4) Together, these results indicate that REDD1 primarily drives endothelial senescence via\natypical NF-κB signaling, while additional NF-κB–independent pathways may also contribute,\nhighlighting its potential multifaceted role in obesity-related vascular dysfunction.\nComment #4: Line 189: The accelerated senescence due to Ad-Redd1 delivery in eNOS KO\nmice suggested that the proposed mechanism of eNOS might not be important. What is the\nrelationship between eNOS and SIRT1? How could REDD1 regulate both of them? Any\nmolecular links between eNOS and SIRT1?\nResponse: (1) We thank the reviewer for raising this important point. Even under control\nconditions (Ad-C), eNOS–/– mice exhibited markedly accelerated vascular senescence\ncompared with WT mice (Supplementary Fig. 7d–i), highlighting the critical role of eNOS in\nvascular homeostasis. Notably, REDD1 delivery in eNOS–/– mice reduced SIRT1 levels and\naggravated senescence, indicating that REDD1 promotes vascular senescence not only via\neNOS loss but also through suppression of SIRT1 expression.\n(2) Mechanistically, REDD1 upregulates miR-155-5p and miR-214-3p, which post-\ntranscriptionally suppress both eNOS and SIRT1 levels (Fig. 3f–i; Supplementary Fig. 4f–k).\nIn the revised Discussion (Lines 401–404), we further clarify the crosstalk between eNOS and\nSIRT1: they form a reciprocal regulatory loop, in which SIRT1 enhances NO production via\neNOS deacetylation (Ref. 40), and NO reciprocally sustains SIRT1 expression (Refs. 16, 24),\ncreating a positive feedback loop essential for vascular homeostasis. REDD1 disrupts this\nSIRT1–eNOS loop, suppressing NO–cGMP signaling and thereby linking metabolic stress to\nendothelial dysfunction.\nComment #1: I co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse: We sincerely appreciate your participation in the co-review process as part of the\nNature Communications initiative. We are grateful for your contribution and for supporting the\ntraining of Early Career Researchers in peer review.\nThis is a comprehensive and well-executed study identifying REDD1 as a central regulator of\nobesity-induced endothelial dysfunction and hypertension through an atypical NF-κB–miRNA–\neNOS/SIRT1 axis. The authors demonstrate the molecular and phenotypic consequences of\nREDD1 activation using an elegant combination of in vitro endothelial models and multiple\ngenetically modified mouse lines (Redd1–/–, Redd1ΔEC, Redd1KKAA, and miR-214-3p–/–). The\nwork provides significant mechanistic insight into how metabolic stressors modulate vascular\naging and function and has clear translational relevance to obesity-related cardiovascular\ndisease.\nMajor Comments:\nComment #1: Limited Vascular Phenotyping in Resistance Arteries: While the study\nthoroughly characterizes REDD1-mediated vascular changes in the aorta, this vessel is\nprimarily a conduit artery and does not directly regulate systemic blood pressure. Since\nresistance arteries (e.g., mesenteric, renal arterioles) are key determinants of vascular tone\nand blood pressure, the absence of functional and molecular assessments in these vessels\nlimits the physiological relevance of the findings. It would significantly strengthen the\nmanuscript to include data on vasomotor responses, NO bioavailability, and eNOS\nexpression/activity in resistance arteries from the same mouse models.\nResponse: (1) We thank the reviewer for emphasizing the role of resistance arteries. To\naddress this, we examined mesenteric arteries after Ad-REDD1 or Ad-REDD1KKAA\nadministration. Ad-REDD1 impaired ACh-mediated vasorelaxation in both thoracic aortic and\nmesenteric arterial rings, whereas Ad-REDD1KKAA had no significant effect (Fig. 4g).\nConsistently, REDD1-dependent impairment was observed in both vessels from DIO-WT but\nnot DIO-Redd1–/– mice (Fig. 6f). Experiments using an NO donor (SNP) and an NOS inhibitor\n(L-NAME) confirmed preserved NO-mediated responses despite reduced eNOS–cGMP\nsignaling (Fig. 6g; Supplementary Fig. 9b).\n(2) We further examined ACh-induced vasorelaxation in mesenteric arterial rings from DIO-\nRedd1fl/fl and DIO-Redd1ΔEC-post mice (EC-specific Redd1 deletion induced after obesity) and\nobserved significantly improved vasorelaxation in DIO-Redd1ΔEC-post mice (Supplementary\nFig. 11h). Although mesenteric arteries could not be assessed in all experimental groups, the\navailable data clearly demonstrate that REDD1 also suppresses eNOS-dependent\nvasorelaxation in resistance arteries. These findings extend the physiological relevance of our\nstudy, showing that REDD1 regulates vascular tone and blood pressure in both conduit and\n23\nresistance arteries. Relevant statements have been incorporated into the Results sections\n(Lines 197–200, 248–251, 290–293).\nComment #2: Kidney Function and Pathology Not Evaluated: Given the central role of the\nrenal vasculature in blood pressure regulation, the manuscript would benefit from analysis of\nrenal function and histopathology. Data on glomerular structure, fibrosis, proteinuria, serum\ncreatinine, and eNOS levels in renal tissue would offer critical insight into whether REDD1\ninfluences hypertension through renal mechanisms in addition to vascular effects.\nResponse: (1) We thank the reviewer for highlighting the importance of assessing renal\nfunction and histopathology in obesity-induced vascular dysfunction. To address this, we\nanalyzed renal structure and function in lean and DIO mice of all genotypes (WT, Redd1–/–,\nRedd1ΔEC, and miR-214-3p–/–). In DIO-WT mice, we observed enlarged glomeruli, abnormal\nrenal architecture, increased renal fibrosis, elevated serum creatinine, and reduced renal\neNOS expression (Fig. 10a–f; Supplementary Fig. 12). These changes were substantially\nameliorated in Redd1–/–, Redd1ΔEC, and miR-214-3p–/– mice. Proteinuria was not assessed\ndue to limited samples.\n(2) These results indicate that endothelial REDD1 activation under DIO contributes to renal\nstructural and functional abnormalities, likely exacerbating blood pressure elevation in addition\nto its vascular effects. Statements reflecting these results have been incorporated in the\nResults sections (Lines 315–323).\nComment #3: Lack of Human Validation: While the preclinical models are robust, no data\nfrom human samples are presented to confirm the upregulation of REDD1 or its downstream\neffectors (miR-155-5p, miR-214-3p, eNOS, SIRT1) in obesity-related hypertension. Inclusion\nof data from human endothelial cells, vascular tissue, or patient serum (even observational)\nwould enhance the translational relevance of the findings.\nResponse: (1) We thank the reviewer for emphasizing the importance of validating our\nfindings in human samples. Although direct analysis of clinical specimens was not performed,\nprevious studies (Refs. 38, 43, 46, 48, 49, 57–62) report molecular changes consistent with\nobservations from this study. Specifically, REDD1, miR-155-5p, and miR-214-3p are elevated,\nwhereas eNOS and SIRT1 are reduced in serum and vascular tissues of obese, hypertensive,\nor metabolically diseased individuals (Refs. 38, 48, 49, 57–62).\n(2) These clinical observations support the translational relevance of the REDD1–atypical NF-\nκB–miRNA axis, suggesting that the mechanisms identified in preclinical models likely\ncontribute to human obesity-associated vascular dysfunction. Statements reflecting this\n24\ndiscussion have been incorporated into the Discussion section (Lines 449–458).\nComment #4: Sex-Specific Differences Not Addressed: All animal experiments appear to\nhave been conducted exclusively in male mice, which overlooks potential sex-specific\ndifferences in REDD1 expression, endothelial aging, and hypertensive responses. Including\nfemale cohorts or, at a minimum, discussing this limitation is recommended to improve the\ngeneralizability of the results.\nResponse: We thank the reviewer for this important comment. All experiments were\nperformed exclusively in male mice to minimize confounding hormonal effects and ensure\nconsistency of metabolic and vascular phenotypes. This has been indicated in the Abstract,\nResults, and Materials and Methods sections. The Discussion (Lines 472–474) now states:\n“First, all experiments were conducted in male mice, and future studies are warranted to\ndetermine whether similar mechanisms occur in females, considering sex-related\nphysiological differences.” These revisions clarify the sex-specific scope of our study and\nhighlight the importance of investigating female models in future work.\nMinor Comments:\nComment #1: DAF-FM DA as a Surrogate for NO Production: The authors use DAF-FM DA\nfluorescence to quantify NO production. While this is a common and convenient method, it\nmeasures bioavailable NO via reaction with NO-derived intermediates (e.g., N O ) and is\n2 3\nconsidered semi-quantitative. This technique does not directly assess NOS enzymatic activity,\nand the fluorescence signal may be influenced by oxidative stress and NO scavenging. To\nmore definitively evaluate eNOS activity, the authors should consider, or at least acknowledge,\nalternative approaches such as [¹⁴C]-L-arginine to [¹⁴C]-L-citrulline conversion assays or EPR\nspectroscopy for detection of authentic NO.\nResponse: (1) We thank the reviewer for highlighting the limitations of using DAF-FM DA\nfluorescence to assess NO production. This method provides an indirect, semi-quantitative\nmeasure of NOS-dependent NO generation, as the fluorescence reflects NO-derived\nintermediates rather than authentic NO. To confirm NOS dependency, endothelial cells and\nvascular tissues were treated with the NOS inhibitor L-NAME, which reduced DAF-FM DA\nfluorescence to basal levels (Figs. 2g, 3b, 5h; Supplementary Figs. 5c, g), indicating that the\nobserved changes primarily reflect NOS activity.\n(2) We have clarified this limitation in the Discussion (Lines 475–478): “Third, eNOS activity\n25\nwas assessed indirectly via DAF-FM DA fluorescence, highlighting the need for more direct\nmethods, such as L-citrulline conversion assays or other approaches for direct NO\nquantification.”\nComment #2: Limitations of Tail-Cuff Blood Pressure Measurement: The authors report\nsystolic, diastolic, and mean arterial pressures using a non-invasive tail-cuff system. While this\nmethod is suitable for SBP measurements, diastolic and MAP values are often unreliable due\nto methodological constraints. Suggested to remove DBP and thus mean BP in all figures.\nTelemetry or intra-arterial catheterisation is the gold standard for accurate, continuous blood\npressure measurement, especially in studies assessing nuanced vascular phenotypes. The\nauthors are encouraged to acknowledge this limitation in the discussion.\nResponse: We thank the reviewer for highlighting the limitations of tail-cuff blood pressure\nmeasurement. Accordingly, DBP and MAP have been removed from all figures, and only SBP\nis now reported. To acknowledge this limitation, we have added the following statement to the\nDiscussion (Lines 474–475): “Second, blood pressure was measured using a tail-cuff system,\nwhich is less precise than telemetry- or catheter-based methods.” These revisions enhance\ntransparency regarding the methodological constraints of our study.\nReviewer #1 (Remarks to the Author):\nThe revised manuscript is much improved and contains a tremendous amount of data in\nsupport of a critical role for REDD1 in mediating endothelial senescence and vascular\ndysfunction in obese mice. The studies are timely and important to the field. While most of my\nprior criticisms are well addressed, some of the key claims made within the manuscript are\nstill not appropriately supported by experimental data. I suggest editing the manuscript to more\naccurately reflect the data or performing additional assessments to provide support for them.\nComment #1: Line 365-369: Without demonstrating the absence of a change in mTORC1\nsignaling, the authors should not claim the changes in senescence with REDD1KKAA are\n“independent of mTORC1 inhibition”. The data are unnecessarily overstated here. While no\nchange in REDD1-TXNIP binding in the REDD1KKAA mutant is supportive of normal protein\nfolding, the concern raised previously that the K219-220 sites are localized to a previously\nidentified “hot-spot” on REDD1 that mediates mTORC1 suppression has not been addressed\nexperimentally by the data.\nResponse: (1) We thank the reviewer for the constructive comment. To directly address the\nconcern regarding mTORC1 signaling, we performed additional experiments assessing S6K\nand 4E-BP1 phosphorylation. Consistent with prior studies, REDD1KKAA failed to inhibit\nmTORC1 activity, as evidenced by restored phosphorylation of S6K and 4E-BP1. Despite this\nlack of mTORC1 inhibition, REDD1KKAA did not significantly affect miR-155-5p, eNOS, SIRT1,\nsenescence markers (SA-β-gal⁺ cells, p53, and p21), or SASP genes (IL1B and IL6), and this\nlack of effect was unchanged by knockdown of Raptor, a key component of mTORC1, with\nonly weak residual senescent features remaining. These data have been incorporated in\nFigure 3jm. Together, these results demonstrate that inhibition of mTORC1 is not sufficient\nto drive endothelial cell senescence, confirming that REDD1-mediated EC senescence occurs\nindependently of mTORC1 inhibition.\n(2) Accordingly, we have revised the Results section to include (Lines 174181): “In contrast,\nREDD1KK219/220AA (REDD1KKAA), which cannot activate NF-κB19,20 but still binds TXNIP\n(Supplementary Fig. 6a) and failed to inhibit mTORC1 activity, as indicated by restored\nphosphorylation of S6K and 4E-BP1, did not significantly affect the levels of miR-155-5p,\neNOS, SIRT1, senescence markers (SA-β-gal⁺ cells, p53, and p21), or SASP genes (IL1B\nand IL6) in HAECs, although some weak senescent features remained (Fig. 3j–m;\ncomponent of mTORC1, did not alter these outcomes, suggesting that REDD1-mediated\ninhibition of mTORC1 is not sufficient to drive EC senescence.”\n(3) We have also revised the Discussion section to clarify the mechanistic interpretation (Lines\n377381) “However, unexpectedly, overexpression of REDD1 promoted vascular senescence\nand hypertension in WT mice, whereas REDD1KKAA, an atypical NF-κB-inactivating mutant that\nretains normal TXNIP interaction and fails to inhibit mTORC1 activity, did not. These findings\nindicate that the protective effects of Redd1KKAA arise from its inability to activate atypical NF-\nκB signaling and are independent of mTORC1 inhibition or protein misfolding.”\nComment #2: It is surprising that only the TNF- mediated response in EC senescence after\n48h do not require REDD1 (i.e., not HG/PA/resistin in Fig. 1). High glucose, palmitate, and\nResistin have previously been shown to enhance secretion of pro-inflammatory cytokines like\nTNF-alpha as well as activate canonical NF-kB signaling. Based on the strong claims that\nREDD1 mediated senescence is mainly independent of IKK, I suggest carrying out\nexperiments to see if resistin/HG/Pal mediated senescence is in fact not dependent on IKK.\nAs it stands now, IKK knockdown is only done with TNF- in Fig S1f, which did not require\nREDD1.\nResponse: (1) We thank the reviewer for this insightful comment. To address this, we\nexamined whether the IKKNF-B axis contributes to EC senescence induced by metabolic\nrisk factors. We found that IKKβ knockdown partially suppressed the increases in SA-β-gal\nactivity induced by palmitic acid, high glucose, or resistin, with weaker inhibitory effects for\nhigh glucose or resistin than for palmitic acid. However, NF-κB p65 knockdown more\neffectively, though not completely, blocked SA-β-gal activity induced by these stimuli.\n(2) The results have been included in Supplementary Figure 1f, and the following texts has\nbeen added to the Results section (Lines 120125); “Given that REDD1 activates NF-B18,\nwe investigated its role in EC senescence. IKKβ knockdown partially suppressed the\nincreases in SA-β-gal activity induced by palmitic acid, high glucose, or resistin, with weaker\ninhibitory effects for high glucose or resistin than for palmitic acid; however, NF-κB p65\nknockdown more effectively, though not completely, blocked SA-β-gal activity induced by\nthese metabolic factors (Supplementary Fig. 1f).”\n(3) Reflecting these results, we revised the graphical abstract (Fig. 10g) and updated the\nrelevant sentences in the Discussion section. (a) Lines 342348: “Collectively, our findings\nshow that REDD1, induced by circulating metabolic risk factors, drives DIO-induced vascular\nsenescence and dysfunction by suppressing eNOS and SIRT1 via atypical NF-κB-responsive\n2\nmiR-214-3p, while these factors also partially engage IKK-dependent NF-κB. REDD1 further\nelevates SASP-related pro-inflammatory cytokines through adipose tissue and endothelial\nsenescence20,22, exacerbating vascular dysfunction via canonical NF-κB activation and\ncrosstalk between atypical and canonical pathways (Fig. 10g).” (b) Lines 352359:\n“Consistently, NF-κB p65 knockdown substantially reversed EC senescence induced by\nmetabolic risk factors or REDD1 overexpression in HAECs, whereas IKKβ knockdown had\nmodest and minor effects, respectively. Pharmacological inhibition of IKKβ partially\nameliorated vascular senescence and dysfunction in both DIO-WT and DIO-Redd1/ mice,\nwith greater efficacy in DIO-WT mice. These results suggest that REDD1 drives EC\nsenescence and vascular dysfunction primarily via atypical NF-B, while metabolic risk factors\nor DIO exert their effects through atypical NF-κB, with additional contribution from the IKKβ-\ndependent pathway.” (c) Lines 377381: “However, unexpectedly, overexpression of REDD1\npromoted vascular senescence and hypertension in WT mice, whereas REDD1KKAA, which\ncannot activate atypical NF-κB but retains normal interaction with TXNIP and fails to inhibit\nmTORC1 activity, did not. These findings indicate that the protective effects of Redd1KKAA arise\nfrom its inability to activate atypical NF-κB signaling and are independent of mTORC1\ninhibition or protein misfolding.” (d) Lines 393396: “Our data show that REDD1KKAA, which\nis unable to activate NF-κB, induces minimal EC senescence and IL6 mRNA expression\ndespite normal TXNIP binding and lack of mTORC1 inhibition, suggesting a modest role for\nNF-κB–independent pathways.”\nRemarks: The authors have conducted additional experiments to address my previous\ncomments. The revised manuscript is now acceptable for publication in Nature\nCommunications.\nResponse: We thank Reviewer #2 for the constructive comments and for acknowledging that\nthe revised manuscript is now suitable for publication.\nRemarks: Authors have addressed my comments.\nResponse: We thank Reviewer #3 for accepting our revised manuscript.\nRemarks: I co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse: We thank Reviewer #4 for their contribution to the peer review of this manuscript\nand for supporting the initiative to recognize Early Career Researchers participating in co-\nreviewing.\nThe authors' reply was helpful and fully addressed my query.\nMinor comments: In figures 4f and 8f, change the y-axis from NOS activity to Aortic NO production.\nResponse: We thank Reviewer #5 for the helpful suggestion. The y-axis in Figures 4f and 8f\nhas been updated from “NOS activity” to “Aortic NO production” accordingly.\n7"}]

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