SREBP1-mediated lipogenesis promotes dedifferentiation and senescence of vascular smooth muscle cells through epigenetic remodeling

Domain: physics
Published: 2025-12-26
Editorial outcome: major_revision

Abstract
Real or simulated microgravity induces a senescence-like modification of carotid artery in both human and animal observations, with the mechanisms not fully elucidated. Here, we aim to elucidate the role of sterol regulatory element-binding protein 1 (SREBP1, encoded by Srebf1) mediated lipogenesis in the process. Pharmacological activation of SREBP1 directly triggers senescence-like transformation in vascular smooth muscle cells (VSMC), while silencing Srebf1 exerts an opposite effect. Mechanistically, SREBP1-mediated lipogenesis upregulates acetyl-CoA pool to increase histone acetylation, modifying the chromatin accessibility which limiting recruitment of SRF/myocardin complexes to CArG boxes of contractile genes and opening the chromatin accessibility of aging genes. Srebf1 knockdown and local delivery of lentivirus or AAV-mediated VSMC specific expressing sh-Srebf1 significantly attenuates the senescence-like transformation of VSMC both in vitro and in vivo. Our findings reveal a previously unrecognized feature of SREBP1-mediated lipogenesis in vascular biology and SM-induced carotid artery remodeling.

Introduction
Space exploration represents a frontier to understand how the human body adapts to extreme environments1,2. Among the various specialties, microgravity exerts a persistent and substantial influence on multiple organs and systems, especially in long-duration space missions3. Astronauts develop several geriatric-like issues, such as bone and muscle mass loss, immune dysfunction, and vascular remodeling, which are also demonstrated in human and animal models designed to simulate microgravity4,5. The carotid artery shows a high sensitivity to various unfavorable impacts6. In both real or simulated microgravity, the carotid artery exhibited a pronounced senescence-like remodeling, including decreased elasticity and increased carotid intima-media thickness (cIMT)2,5,7–10. Vascular smooth muscle cells (VSMC) are the primary cells that form the tunica media11. Differentiated VSMC exhibit a quiescent, contractile phenotype and express genes involved in contraction, such as smooth muscle actin (Acta2, encoding α-SMA), smooth muscle protein 22-α (Tagln, encoding SM22α), smooth muscle myosin heavy chain (Myh11, encoding MYH11), and calponin (Cnn1, encoding CNN1)12,13. With a notable plasticity, VSMC turn to be reversibly dedifferentiated and transformed to suit the vascular growth, remodeling, and repair upon environmental stimulates12–15. Dysregulated transformation of VSMC comes along with vascular pathologies in aging, atherosclerosis and aortic aneurysm16. For now, the characteristics and molecular mechanisms behind the phenotypic transformation of VSMC in simulated microgravity are still not fully clarified.

Lipids are key macromolecules engaged in biological activities from cell membrane construction, energy storage, to signaling transduction17. Recent studies indicate significant modification of lipid metabolism in senescence or age-related diseases18,19. Sterol regulatory element binding protein (SREBP, encoded by Srebf) acts as a key transcription factor (TF) controlling the expression of genes in charge of lipid production, degradation and conversion20,21. It has been well known that SREBP1 mainly contributes to fatty acid (FA) synthesis, while SREBP2 to the synthesis and uptake of cholesterol (CL)22. Recent studies unveiled the negative effects of SREBP cleavage-activating protein (SCAP, encoded by Scap) in atherosclerosis23,24. Some other genes, such as Fasn (encoding fatty acid synthase) and Hmgcr (encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase), played a key role in VSMC related diseases25,26.

Lipid metabolites work through multiple downstream biological processes. Among them, epigenetic modifications of chromatin have provided novel insights to etiological exploration of diseases, especially those cannot be fully explained by genetic mechanisms. Recent studies revealed the connection between acetyl-CoA (Ac-CoA), a key metabolite coordinating energy metabolism of different pathways involving lipid homeostasis and chromatin adaptation27,28. For doing this, histone acetylation and deacetylation are modified, which regulate chromatin accessibility by facilitating and blocking TFs access, respectively. This would change the electrostatic interactions between DNA and the histones that assemble the nucleosome29. Accumulating evidence links histone acetylation to aging-related vascular disorders30. The increase in histone-H3 lysine-27 (H3K27) acetylation contributes to vascular senescence by opening up stiffness gene promoters31. Histone deacetylase inhibitors promote histone hyperacetylation accelerating vascular calcification in preclinical studies32,33. Given the findings depicted above, it was hypothesized that the Ac-CoA pool harmonized the lipid homeostasis and gene expression in carotid artery, which regulated the VSMC phenotypic transformation and senescence-like remodeling in response to simulated microgravity.

In this work, we discover an accumulation of Ac-CoA and the following adjustment of SREBP1-mediated lipogenesis. Furthermore, an altered chromatin accessibility is demonstrated, playing a crucial role in the vascular aging process.

Results
Simulated microgravity leads to senescence-like transformation of VSMC in carotid artery
To efficiently simulate the effects of microgravity in vivo, the hindlimb unloaded tail suspended (HU) rat model was established as previously described (Supplementary Fig. S1A)34. Compared with control (CON) rats, 4 weeks of HU rats evinced classical manifestations of microgravity, including atrophy of soleus muscle fibers and natural body weight gain with reduced ratio of soleus to body weight, verifying the efficacy of the model (Supplementary Fig. S1B–E). Pathological staining and vascular ultrasound test revealed a senescence-like structural remodeling in carotid artery of HU rats, which involved a deranged media, broken elastin, and thickened cIMT, but normal lumen diameter (Fig. 1B–D and Supplementary Fig. S1F, G). We further confirmed the downregulated expression of VSMC’s contractile genes (Acta2, Tagln, Myh11, Cnn1), the upregulated expression of aging genes (Cdkn1a, Cdkn2a, Tp53) or senescence-associated secretory phenotype (SASP) genes (Il6, Ccl2) by RT-qPCR, western blotting, immunofluorescence, immunohistochemistry, and higher activity of β-Galactosidase in the carotid artery of HU rats (Fig. 1E–H and Supplementary Fig. S1H–L). Notably, the increased P21 (encoded by Cdkn1a) signals was mainly distributed in VSMC (marked by α-SMA) (Supplementary Fig. S1H, 1H).

This observation prompted us to examine whether there was a similar change in simulated microgravity cell models. To simulate microgravity in vitro, we generated a 2-dimensional rotation (SM model) of mouse aortic smooth muscle cells (MOVAS) as previously described (Supplementary Fig. S2A, B)35. Firstly, the SM cell models exhibited a senescence-like transformation consistent with the HU model, including the downregulation of contractile genes and upregulation of aging or SASP genes (Fig. 1I, J). Moreover, as shown in Fig. 1K, L and Supplementary Fig. S2C, SM treated MOVAS displayed a higher intensity of SPiDER-βGal fluorescence, higher activity of β-Galactosidase, and bigger cell size. In addition, the contractility of MOVAS in the SM group was lower than in CTRL (Fig. 1M). Similarly, SM treated human aortic smooth muscle cells (HASMC) also exhibited senescence-like transformation (Supplementary Fig. S2D–F). However, human umbilical vein endothelial cells (HUVEC), a classic endothelial cell model, had no significant senescent signals following SM treatment (Supplementary Fig. S2G–I).

Moreover, the transcriptomic features of the carotid artery from the HU model (CON as control) and the SM cell model (CTRL as control) were analyzed by bulk RNA-seq. Among 5328 different expression genes (DEGs) of HU rats, 3916 were upregulated, while 1412 were downregulated (Supplementary Fig. S3A–C). Among 961 DEGs of SM cells, 530 were upregulated, while 431 were downregulated (Supplementary Fig. S3D–F). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that the DEGs were enriched in lipid metabolism, cardiovascular disease, gene replication and repair, as well as cell growth and death in both HU or SM models (Supplementary Fig. S3G, H). Gene Ontology (GO) based Gene Set Enrichment Analysis (GSEA) demonstrated that the processes related to VSMC biology, such as differentiation, proliferation, and contraction, were significantly inhibited in HU or SM model (Fig. 1N). Moreover, compared with CTRL, SM treated MOVAS exhibited sparser cell density, larger cell size (Supplementary Fig. S2J), lower Ki-67 expression (a marker of cell proliferation) (Supplementary Fig. S2K), and higher apoptosis signaling (Supplementary Fig. S2L). Notably, cells that were shed following SM treatment exhibited stronger apoptotic signaling and higher expression of senescent marker genes (Supplementary Fig. S2M, N).

Together, these findings indicate that the SM leads to a senescence-like transformation of VSMC in the carotid artery.

Simulated microgravity induces lipid accumulation in VSMC
To further investigate the potential mechanisms of senescence-like transformation of VSMC caused by simulated microgravity, we performed combining analysis for the transcriptomes of HU and SM models. Based on KEGG-associated GSEA analysis, multi-pathways involved in lipid metabolism were enriched in the 47 co-activated pathways (NES>1.0) both in HU and SM models, such as fatty acid metabolism, fatty acid biosynthesis, or steroid biosynthesis (Supplementary Fig. S4A and Fig. 2A). Multi-pathways involved in cytoskeleton and cell contraction were enriched in the 16 co-inhibited pathways (NES<−1.0), such as TGF-beta signaling pathway, ECM-receptor, and vascular smooth muscle contraction (Supplementary Fig. S4B, C). In addition, heatmap results showed that both HU and SM models led to the activation of cholesterol and fatty acids synthesis pathways, which represented the key pathways for the intracellular lipid homeostasis (Fig. 2B). Further GO analysis indicated that DEGs were also enriched in multi-lipid metabolism-related pathways, both in HU and SM models (Supplementary Fig. S4D, E).

This observation prompted us to examine whether there was a dyshomeostasis of lipids in SM cells and HU rat models. Firstly, transmission electron microscopy (TEM) results disclosed an accumulated lipid droplets in SM MOVAS (Fig. 2C). This was then confirmed by the staining of Nile Red and BODIPY, two lipophilic fluorescent probes (Fig. 2D, E). Furthermore, rat primary carotid artery smooth muscle cells (RCA-SMCs) were isolated to elucidate whether lipid accumulation remained in SM treated primary cells (Supplementary Fig. S5A, B). Consistent with MOVAS, SM treatment significantly increased the content of fatty acids (FA) and triglyceride (TG), leading to lipid accumulation in RCA-SMCs (Supplementary Fig. S5C–E). SM-treated HASMC also exhibited lipid accumulation (Supplementary Fig. S5F). In addition, lipidomics was performed to profile the lipid state of the carotid artery (Supplementary Fig. S6 and Supplementary Data 1). We found an abundant distribution of lipids in the carotid artery of rats (Supplementary Fig. S6A). Among the 21 classes (1097 species), TG, FA, and phosphatidylcholine (PC) were the top three in the carotid artery (>1 mg/g) (Figure S6B). As compared with CON, HU rats showed a higher lipid content of multi-lipid species in carotid artery (Fig. 2F and Supplementary Fig. S6C, D), including FA, cholesterol ester (ChE), monoglycerides (MG), diglycerides (DG), and TG (Fig. 2G–K).

Taken together, these data demonstrate that the SM induces lipid accumulation in VSMC of the carotid artery.

Simulated microgravity activates SREBPs mediated lipogenesis
As a TF, SREBPs activate the expression of numerous genes essential for lipid homeostasis20. To explore its role in lipid accumulation triggered by SM, we performed a GSEA based activated TF enrichment analysis (NES>1, normalized P<0.05) for the transcriptomics of HU and SM models. As shown in Fig. 3A, 75 and 102 TFs were significantly enriched in HU and SM models, respectively. There were 21 of these TFs co-enriched in both HU and SM models. Intriguingly, SREBP1 (a crucial TF in lipid biogenesis) was listed among these 21 co-enriched TFs (Fig. 3A). GSEA results indicated the activation of SREBP1 in both HU and SM models (Fig. 3B). Because of the absence of SREBP2 in candidate TFs, it was not listed among the co-enriched 21 TFs. However, as SREBP2 shares a similar DNA-binding motif with SREBP1, it might also be activated during SM (Supplementary Fig. S7A, B).

Then, we confirmed the upregulated expression of SREBP1 and SREBP2 by immunohistochemistry and immunofluorescence in the carotid artery of HU rats (Fig. 3C and Supplementary Fig. S7C). Notably, the increased SREBP1 signals was mainly distributed in VSMC (marked by α-SMA) (Supplementary Fig. S1H and Fig. 3D). Western blot further demonstrated the increased protein expression of precursor-SREBPs (p-SREBPs) and nucleic-SREBPs (n-SREBPs) in HU rats compared with CON (Fig. 3E). Using RT-qPCR assays, the upregulation of Srebfs (SREBPs encoding genes), Scap (activator of SREBPs), and representative targets of SREBPs was discovered, strongly indicating the activation of SREBPs in HU rats (Fig. 3F). In SM-treated VSMC models, protein levels of p-SREBPs and n-SREBPs were also increased (Fig. 3G and Supplementary Fig. S7D). In addition, the increasing levels of nuclear-distributed SREBPs and transcripts of SREBPs target genes also demonstrated the activation of SREBPs in SM treated MOVAS or HASMC (Fig. 3H, I and Supplementary Fig. S7E, F). However, HUVEC showed no significant changes of SREBPs after SM treatment (Supplementary Fig. S7G). Given to the well-established crucial role of mTOR signaling in activating SREBP1, mechanotransduction, and cell senescence20,36–40, we further confirmed the activation of the mTOR pathway, which was demonstrated by the upregulation of p-mTOR/mTOR and p-AKT/AKT (key kinases bridging mechanical signaling to mTOR activation).

All these results indicate that the activation of SREBPs contributes to the lipid accumulation of the carotid artery and VSMC induced by SM.

SREBP1 directly promotes the senescence-like transformation of VSMC by activating lipogenesis
Given the nonidentical roles of SREBP1 and SREBP2 in lipid homeostatic regulation, we tried to examine the potential differences in their contribution to VSMC biology. We firstly analyzed the expression correlation between the SREBPs target genes and contractile (or aging) genes in the transcriptome dataset of human carotid artery from atherosclerotic and healthy individuals (GSE100927)41. As shown in Fig. 4A, SREBPs-targets were positively correlated with aging genes and negatively correlated with VSMC contractile genes. Intriguingly, these correlations were mainly attributed to SREBP1-targets but less to SREBP2-targets (Supplementary Fig. S8A–C). To further elucidate whether such correlations exist in VSMC at the single-cell level, we analyzed the average expression of VSMC contractile, aging, and SREBP1-related genes across different subgroups of VSMC in the carotid arteries from atherosclerosis patients (GSE253903) (Supplementary Fig. S8D–G). Intriguingly, the VSMC subgroup with higher expression of contractile genes exhibited lower expression of SREBP1-related and aging genes. In contrast, the VSMC subgroups with lower expression of contractile marker genes showed higher expression of SREBP1-related and aging genes (Fig. 4B). In addition, H2O2, a canonical senescence-inducer, could directly upregulate the expression of SREBP1 and its target genes, while also induce intracellular lipid accumulation in VSMC (Supplementary Fig. S9A–F), which was consistent with previous studies42.

Then, we generated Srebf1 (or Srebf2) knockdown MOVAS by siRNA transient transfection (Supplementary Fig. S10A–D). Surprisingly, compared with siCtrl, si-Srebf1 exhibited greater contractile function, which was reflected in higher expression of contractile genes and stronger contractility in MOVAS (Fig. 4C, D and Supplementary Fig. S10E, F). In contrast, si-Srebf2 exhibited lesser contractile function, which was reflected in lower expression of contractile genes and weaker contractility in MOVAS (Fig. 4C, D and Supplementary Fig. S10E, F). To determine whether SREBP1 activation was directly responsible for the senescence-like transformation of VSMC, we performed a targeted agonist (LXR-623) treatment to activate SREBP1 in MOVAS or RCA-SMCs (Fig. 4E). Consistent with previous reports, we found that LXR-623 activated SREBP1 but not SREBP2, demonstrated by increased p-SREBP1, n-SREBP1, as well as its target genes’ expression both in MOVAS, RCA-SMCs, and HASMC (Fig. 4F and Supplementary Fig. S11A–G). We further performed RNA-seq and found 1267 genes were upregulated, while 2278 genes were downregulated in LXR-623 relative to DMSO treated MOVAS (Supplementary Fig. S12A–C). KEGG analysis indicated that the DEGs were enriched in lipid metabolism, cardiovascular disease, gene replication and repair, as well as cell growth and death, which was consistent with HU or SM models (Supplementary Figs. S12D, S3G, H). Notably, as shown in Fig. 4G, H, SREBP1 was significantly enriched as one of the co-activated TFs in all three transcriptomes (CON vs. HU; CTRL vs. SM; DMSO vs. LXR-623). GO analysis further verified the enrichment of DEGs in pathways related to lipid biosynthetic and metabolic processes (Supplementary Fig. S12E). Meanwhile, the heatmap revealed that LXR-623 treatment directly activated FA synthesis but not CL metabolism, suggesting the SREBP1 but not SREBP2 being the target of LXR-623 (Fig. 4I). In addition, MOVAS exhibited lipid accumulation after LXR-623 treatment, demonstrated by the staining of BODIPY (Fig. 4J). These results were further supported by the observation of extensive lipid droplets in LXR-623 treated MOVAS by TEM (Fig. 4K). The LXR-623 induced lipid accumulation was also confirmed in HASMC (Supplementary Fig. S11H).

We next determined the pathological role of LXR-623 for VSMC biology. Compared with DMSO, LXR-623 treated VSMC showed weaker contractility and lower expression of contractile genes (Fig. 4L, M and Supplementary Fig. S11I–L). GO based GSEA results demonstrated that vascular associated smooth muscle cell contraction and differentiation were depressed by LXR-623 (Fig. 4N and Supplementary Fig. S12F). Moreover, LXR-623 directly triggered cell senescence, which was indicated by a higher intensity of SPiDER-βGal fluorescence, higher activity of β-galactosidase, and upregulation of aging and SASP genes (Fig. 4O, P and Supplementary Fig. S11M–O). Furthermore, mTOR signaling was also activated by LXR-623 treatment, which was consistent with SM treatment (Supplementary Figs. S11P, Q, S7G, H).

Taken together, these data reveal that the targeted activation of SREBP1 directly promotes VSMC’ senescence-like transformation.

Upregulated Ac-CoA pool contributes to SREBP1-activation triggered senescence-like transformation of VSMC
It has been well established that the Ac-CoA pool takes a central role in energy production, lipid metabolism, and gene expression (Fig. 5A)27. Therefore, we investigated whether SREBP1 activation disturbed the balance of the Ac-CoA pool in VSMC. We observed that the Ac-CoA levels were significantly enhanced in MOVAS after both LXR-623 and SM treatments, with the citrate, a key substate of endogenous Ac-CoA synthesis, being significantly decreased (Fig. 5B and Supplementary Fig. S13A). Notably, the increased Ac-CoA levels were predominantly localized in the cytoplasm and nucleus, while no significant changes were observed in the mitochondrial Ac-CoA levels (Supplementary Fig. S13B, C).

Cellular and nuclear Ac-CoA pool has been reported manipulating the regulation of gene expression by protein’s acetylation, particularly for histones27. In the present study, cellular protein acetylation was amplified substantially by LXR-623 or SM treatment (Fig. 5C, D and Supplementary Fig. S13D). We then performed IP-MS to characterize the acetylated protein profile (anti-Pan-acetylation) in LXR-623 or SM treated MOVAS (Fig. 5E, F and Supplementary Fig. S13E, F). Among the 101 (122 in SM) identified proteins, the 60 proteins in the LXR-623 group (105 in SM group) exhibited hyperacetylation. And these hyperacetylated proteins were significantly enriched in the regulation of gene expression terms, such as nucleosome, chromatin DNA binding, and nucleosome assembly (Fig. 5G, Supplementary Fig. S13G and Supplementary Data 2, 3). These pathways are intimately linked to histone H3 hyperacetylation, which was further confirmed by the increased level of H3K9Ac, H3K18Ac, and H3K27Ac (representative acetylated histone sites) in LXR-623 or SM treated group, compared with DMSO or CTRL (Fig. 5H, Supplementary Fig. S13H–J). Similarly, the increased levels of H3K27Ac were also confirmed in VSMC of HU rat model (Supplementary Fig. S13K).

To determine whether the increased Ac-CoA pool directly plays a role in senescence-like transformation of VSMC, we employed sodium acetate (NaAc, an exogenous Ac-CoA supplements) and Deacetylase Inhibitor Cocktail (DaIC, indirectly increasing endogenous protein acetylation) to the MOVAS (Fig. 5I). As expected, both the Ac-CoA levels and acetylated protein abundance were elevated by these two supplements (Supplementary Fig. S14A–D). In addition, the Acta2 expression was downregulated, and the Cdkn1a was upregulated significantly. The senescence-like transformation of VSMC induced by LXR-623 was further exacerbated upon treatment with NaAc or DaIC, characterized by increased acetylation of H3 and upregulation of P21, while the contractile phenotype was suppressed (Fig. 5J–M, Supplementary Fig. S14F, G). In addition, supplementation with NaAc partially offset the effects of Srebf1 knockdown, including the mitigation of intracellular lipids, H3 acetylation, and the expression of Acta2 and Cdkn1a (Supplementary Fig. S14H–J).

This observation prompted us to examine whether Acly (encoding ATP-citrate lyase) and Acss2 (encoding Ac-CoA synthetase 2) were upregulated, which are two targets of SREBP1 and key enzymes catalyzing the Ac-CoA biosynthesis from endogenous citrate (ACLY) and from exogenous acetate (ACSS2). Intriguingly, Acly and Acss2 were both upregulated in HU rats, SM or LXR-623 treated VSMC (Supplementary Fig. S15A–C). However, the expression of Acss1, which catalyzes the synthesis of mitochondrial Ac-CoA, exhibited no significant changes, in line with the stable levels of mitochondrial Ac-CoA (Supplementary Figs. S15D, E, S13B, C). We further generated Acly or Acss2 knockdown MOVAS by siRNA transient transfection to determine whether ACLY or ACSS2 were involved in the senescence-like transformation mediated by SREBP1 activation induced by SM (Supplementary Fig. S15F–I). Notably, both Acly or Acss2 knockdown significantly alleviated SM-induced lipid accumulation and cellular senescence in VSMC, which indicated by lower signals of BODIPY, SPiDER-βGal, and P21 expression (Supplementary Fig. S15J–L).

Together, these results demonstrate that the upregulated Ac-CoA pool contributes to the SREBP1-activation triggered senescence-like transformation of VSMC.

Upregulated Ac-CoA pool accelerates the phenotypic transformation of VSMC by reshaping chromatin accessibility
Since SREBP1 activation upregulated the intracellular Ac-CoA pool and resulted in an increase of histone acetylation, we next examined whether chromatin accessibility was involved in senescence-like transformation of MOVAS. We performed ATAC-Seq by Tn5 transposase in MOVAS treated with DMSO, LXR-623, DMSO+DaIC, or LXR-623+DaIC (Fig. 6A and Supplementary Fig. S16A). Totally, 69795 peaks were identified which lying across promoter (32.5%), exon (2.8%), intron (32.67%), 5’ UTR (0.08%), 3’ UTR (1.2%), downstream (0.15%), and distal intergenic (30.6%) (Supplementary Fig. S16B).

We further divided the genes into eight clusters (cluster 1-8) according to the signals of promoter peaks (22710 peaks of 17703 genes) in each group (Fig. 6B and Supplementary Fig. S16C). Genesets of cluster-5, -6, and -8 attracted our attention. Cluster-5 represented genes with condensed chromatin accessibility after LXR-623 treatment, which was more condensed after DaIC supplement. Based on KEGG analysis, cluster-5 genes were enriched in pathways of cytoskeleton, including MAPK signaling pathway, cytoskeleton in muscle cells, and regulation of actin cytoskeleton (Fig. 6B). Further GO analysis indicated that cluster-5 were involved in vascular remodeling (angiogenesis; artery development; etc), VSMC dedifferentiation (muscle cell differentiation; positive regulation of cell motility; and migration; etc), and cytoskeleton (regulation of Rho protein signal transduction; etc) (Fig. 6C). Conversely, cluster-6 and -8 represented genes with opened chromatin accessibility after LXR-623 treatment which was more opened after DaIC supplement (Fig. 6B). Based on KEGG and GO analysis, these two clusters were significantly enriched in pathways involving epigenetic remodeling (ATP-dependent chromatin remodeling, chromatin remodeling, etc), aging (cellular senescence, cell cycle, apoptosis, etc), and lipid metabolism (lipid and atherosclerosis, lipid biosynthetic process, acyltransferase activity, etc) (Fig. 6B, D and Supplementary Fig. S16D). Intriguingly, addition of the LXR-623 or DaIC downregulated the chromatin accessibility of key contractile genes, while upregulating the chromatin accessibility of SREBP1-related genes and aging genes (Fig. 6E). For Acta2, the chromatin accessibility was partially blocked by LXR-623 or DaIC. A synergistic effect was uncovered if supplying DaIC after LXR-623 treatment (Fig. 6F). In contrary, the chromatin accessibility of Cdkn1a was opened after treating with LXR-623 or DaIC. A similar synergistic effect was also present when the two supplements were employed (Fig. 6F).

To gain insight into the underlying mechanisms of VSMC’ dedifferentiation triggered by the upregulated SREBP1 activation, we performed TFs enrichment analysis and compared it with that of RNA-seq (Fig. 6G). TFs enrichment analysis suggested that serum response factor (SRF), as one of the three hub TFs, might be involved in the contractile fate inhibition of VSMC (Supplementary Fig. S16E). Based on GSEA analysis, SRF was also the hub TFs contributing to the genes’ downregulation of three transcriptomics performed in this study (CON vs. HU; CTRL vs. SM; DMSO vs. LXR-623) (Supplementary Fig. S16E and Fig. 6H, I). Further results indicated that LXR-623 or SM treatments inhibited the nuclear translocation of SRF and MYOCD (a transcriptional coordinator of SRF), which might contribute to the functional inhibition of SRF (Fig. 6J and Supplementary Fig. S16F, G). In addition, the downregulated SRF targets were mainly enriched in actin cytoskeleton organization, muscle structure development, and response to mechanical stimulus, which contributed to the inhibition of VSMC’ differentiated fate caused by SREBP1 activation and simulated microgravity (Fig. 6K and Supplementary Fig. S16H).

Taken together, these data reveal that upregulated Ac-CoA pool triggers VSMC’ senescence-like transformation by reshaping chromatin accessibility.

Knockdown of Srebf1 alleviates senescence-like transformation of VSMC induced by simulated microgravity
As the SM upregulated the Ac-CoA pool and reshaped chromatin accessibility by activating SREBP1, we investigated whether downregulating Srebf1 using lentivirus (Lv) exerted protection on VSMC senescence and vascular remodeling (Fig. 7A, G). First, we assessed the effects of Lv-mediated Srebf1 knockdown (Lv-sh-Srebf1) on RCA-SMCs. Lv-sh-Srebf1 treatment significantly downregulated the expression of Srebf1, Acly, Acss2, and aging genes, as well as the protein levels of ACLY, ACSS2, P21, and H3 acetylation (Supplementary Fig. S17A–D). Knockdown of Srebf1 alleviated the upregulation of Ac-CoA levels, Srebf1, Acly, and Acss2 genes triggered by SM (Fig. 7B, C). In addition, Srebf1 knockdown partially alleviated the senescence-like transformation of RCA-SMCs due to SM treatment, which was manifested in the restoration of the cell contractility and expression of H3 acetylation, P21, and α-SMA (Fig. 7D–F).

To achieve local intervention of SREBP1 in carotid artery, we used Lv-sh-Srebf1 loaded by Laponite hydrogels (Fig. 7G, H)43. As shown in Supplementary Fig. S18A–D, Laponite hydrogels mixed with Lv effectively introduced the target sequences into VSMC of the carotid artery. Further ultrasound analysis revealed that Lv-sh-Srebf1 mitigated the HU-induced cIMT thickening but not affecting the diameter of the lumen (Fig. 7I, J and Supplementary Fig. S18E, F). Srebf1 knockdown alleviated HU-induced senescence-like media derangement and elastin break (Fig. 7K). Notably, the Ac-CoA levels were significantly increased in the carotid artery of HU rats compared with CON, which was partially reduced by Lv-sh-Srebf1 transfection, which was consistent with RCA-SMCs (Fig. 7L). In addition, in line with attenuated structural remodeling, Lv-sh-Srebf1 treatment partially restored the disordered transcription of Srebf1, Acta2, Tagln, Cdkn1a, and Tp53 as well as the expression of P21 and Pan-Ac in the carotid artery treated with HU (Fig. 7M, N). To achieve local VSMC-specific intervention of SREBP1 in mice CA, we used AAV-Tagln-sh-Srebf1 loaded by Laponite hydrogels (Supplementary Fig. S19A, B). As shown in Supplementary Fig. S19C, Laponite hydrogels mixed with AAV effectively introduced the target sequences into VSMC of the mice carotid artery. VSMC-specific Srebf1 knockdown alleviated HU-induced senescence-like derangement and elastin break of media (Supplementary Fig. S19D). Further ultrasound analysis revealed that AAV-Tagln-sh-Srebf1 mitigated the HU-induced cIMT thickening but not affecting the diameter of lumen (Supplementary Fig. S19E–H). Notably, VSMC-specific Srebf1 knockdown also partially recovered the disordered expression of SREBP1 related genes, aging genes, and contractile genes (Supplementary Fig. S19I–L) in HU mice carotid artery, which was consistent with Lv-sh-Srebf1-treated rats.

Overall, these findings suggest that Srebf1 knockdown imposed functional and morphological benefits on SM induced senescence-like remodeling of carotid artery, highlighting the pathological essence and potential therapeutic value of SREBP1.

Methods
Animal experiments
Male Sprague-Dawley male rats (6~7 weeks old, body weight: 200±10g) and C57BL/6 (6~8 weeks old) male mice were obtained from laboratory animal center of Fourth Military Medical University, and individually caged within a 12:12 h light/dark cycle at 23±1°C with standard lab chow and water available free to access. All animal experiments in this study were conducted using male rats/mice exclusively to eliminate potential confounding effects of sex hormones on experimental outcomes, consistent with standard practices in this field of research. Special attention was paid to animal welfare and the minimization of suffering. All animal experiments were followed the rules of the American Association for the Accreditation of Laboratory Animal Care International. All procedures were approved by the Animal Care Committee of the Fourth Military Medical University accredited by AAALAC International (No. 20210429). We have complied with all relevant ethical regulations for animal use.

Hindlimb unloading (HU) models
After 1 week (W) of accommodation, the rats or mice were randomly assigned to control (CON) and HU group. HU was achieved by maintaining the animals in a 30° head-down tilt position using a tail harness, with the fully extended hindlimbs 0.5 cm above the floor79,80. HU duration was 4 W. The CON groups were synchronized timely to HU group. The ratio of left soleus wet weight to body weight (SW/BW, mg/g) was calculated to verify the effectiveness of HU model.

Ultrasound imaging
A Vevo 3100 LAZR ultrasound system (FUJIFILM Visual Sonics, Canada) was used for vascular ultrasound imaging. Rats or mice were anaesthetized by 1.5–2% isoflurane and placed on the platform. Luminal diameter and vessel wall thickness were measured by M-mode ultrasonography at carotid artery.

Morphological analysis
H&E, Masson’s trichrome, and Verhoeff’s Van Gieson staining (EVG) staining were employed for further morphological analysis. After euthanasia, rats or mice were transcardially perfused with PBS followed by 4% PFA. The tissues were isolated and embedded in paraffin. Successive 8 μm sections were cut for further experiments. These sections were stained with H&E, Masson’s trichrome, and EVG to evaluate morphology (Servicebio). All the graphs were taken by the Olympus VS200.

In situ transfection of the carotid arteries by lentivirus or AAV loaded in Laponite hydrogels
Lentivirus encoding mCherry protein or sh-Srebf1 was constructed by General Biol Co. Ltd. (Anhui, China) or Tsingke Biotechnology Co., Ltd. (Beijing, China). 50 μL lentivirus (titer: 1.5×108 TU/mL) were mixed with 50 μL Laponite hydrogels, and 100 μL of the mixture was applied locally to the adventitia of the carotid arteries of the rats. In Supplementary Fig. S18B, 14 days after the surgery, the rats were euthanized, and the carotid artery were separated from surrounding tissues, harvested, and observed with the IVScope 8200 In Vivo Imaging System (Clinx, Shanghai, China). Similarly, AAV2 encoding mCherry protein or Tagln-mir30-sh-Srebf1 was constructed by General Biol Co. Ltd. (Anhui, China). 25 μL lentivirus (titer: 3×1012 Vg/mL) were mixed with 25 μL Laponite hydrogels, and 50 μL of the mixture was applied locally to the adventitia of the carotid arteries of the mice.

Cell culture and transfection
MOVAS, HASMC, and HUVEC cells are obtained originally from ATCC. MOVAS were maintained at 37°C with 95% air and 5% CO2 in DMEM (Sigma) supplemented with 10% fetal bovine serum (FBS, Gibco), streptomycin (100 μg/ml) and penicillin (100 IU/ml) (Gibco). HASMC were maintained at 37°C with 95% air and 5% CO2 in Smooth Muscle Cell Medium (ScienCell, Cat. #1101). HUVEC were maintained at 37°C with 95% air and 5% CO2 in Endothelial Cell Medium (ScienCell, Cat. #1001). Before treatment, cells were seeded in T25 plates, 6-well plates or 20 mm confocal dishes and cultured for 12 h.

Rat primary carotid artery smooth muscle cells (RCA-SMCs) were isolated from the carotid artery of adult male Sprague Dawley rats. Anaesthetize the rats by 2% isoflurane, dissect the neck and excise their carotid artery. After removing the adventitia, the carotid artery tissues were cut open and scraped off the intima gently. Then, the carotid artery were washed with cold DPBS (Sigma), minced into ~1 mm3 pieces, and digested using 1 mg/ml collagenase Ⅰ (BioFROXX) at 37°C in a water bath for ~30 min. After digestion, the supernatant was collected and mixed with DMEM (Gibco). Cells were collected by centrifuging at 500×g for 5 min and further resuspended in DMEM supplemented with 10% FBS (Gibco), streptomycin (100 μg/ml) and penicillin (100 IU/ml) (Gibco). The purity of RCA-SMCs was tested by immunofluorescence staining of α-SMA (Proteintech, Cat. #14395-1-AP, 1:200). Isolated RCA-SMCs were maintained in low-glucose DMEM (Gibco) containing 10% FBS (Gibco), and cells at passages 4 to 8 were used for further experiments.

For gene knockdown, MOVAS were transfected with siRNA by TSnanofect V2 transfection Reagent (Cat. #TSV405, Tsingke Biotechnology Co., Ltd. Beijing, China) according to the manufacturer’s protocol. The corresponding sequences are listed in Supplementary Table 2.

For Srebf1 knockdown, RCA-SMCs were infected with lentivirus (Lv) carrying negative control (shCtrl) or knockdown of rat Srebf1 gene (sh-Srebf1) at an MOI of 100 from Tsingke Biotechnology Co., Ltd. (Beijing, China). The corresponding sequences are listed in Supplementary Table 2. After puromycin screening, RCA-SMCs was used for further assays.

All cells were maintained in a humidified 5% CO2 incubator at 37°C.

Two-dimensional rotation-simulated microgravity cell models (SM)
To simulate microgravity, we used a two-dimensional (2D) clinostat, which was designed and provided by the China Astronaut Research and Training Center (Beijing, China)35,81. Rotation causes a gravity vector that is not recognizable by cells. Therefore, the device prevents the cells from feeling gravity. In the present study, cells were incubated in T25 culture flasks or plated on confocal dishes for following experiments. After cell adhesion, flasks or flasks mounted with coverslips were filled up with culture medium to prevent the presence of air bubbles. The dishes were fixed carefully to the rotating panel of the clinostat system, and rotated at a constant speed for 48 h to simulate microgravity (0.01 g). Cells without rotation (1 g) were cultured in the same manner as the control group.

Senescence β-Galactosidase staining
Senescence β-Galactosidase was stained by using a SA-β-gal detection kit (Beyotime Biotechnology, Shanghai, China). The cells or tissues were immersed in the staining solution and incubated at 37°C for 12 h. Blue staining were suggested to be SA-β-gal-positive signals.

Cell contraction assay
A Cell Contraction Assay Kit (CBA-021) was purchased from Cell Biolabs, Inc. (San Diego, CA, USA). The contractile ability of the cells was evaluated according to the manufacturer’s instructions. Cells were harvested and resuspended in culture medium. A collagen lattice was prepared by mixing the cell suspension and ice-cold collagen gel solution in a volume ratio of 1:4. Then, 0.1 mL of the cell-collagen mixture (containing 104 cells) was added to a 96-well plate and incubated for 1 hour at 37°C. After collagen polymerization, 0.1 mL of culture medium was added on top of each collagen gel lattice. 24 h later, the plates were scanned. For data analysis, the area of the gel in each well was analyzed by Fiji Image J (NIH, Bethesda, MD, United States).

Cell apoptosis assay
Annexin V-FITC Apoptosis Detection Kit (Beyotime, Cat. #C1062S) was used to stain apoptotic cells as per the manufacturer’s instructions. Briefly, cells were seeded in T25 plates and treated with SM model for 48 h. Cells were trypsinized, harvested and stained with Annexin-V and PI for 15 min at RT in assay buffer. Annexin-V stained cells were centrifuged and washed with assay buffer. Next, cells were measured by CytoFLEX (Beckman Colter Life Sciences). Data was analyzed with FlowJo v.10 software (BD Life Sciences).

Transmission electron microscopy (TEM)
After washing with PBS, cell samples were fixed in 2.5% glutaraldehyde and 0.1 M sodium cacodylate buffer at 4°C overnight. All the samples were embedded and subjected to ultramicrotomy. Transmission electron microscope images were captured by JEM-1400 (Japan Electron Optics Laboratory) and iTEM 5.2 (EMSIS).

Confocal microscopy
After specific treatment, cells in confocal dishes were washed by pre-warmed DPBS twice and followed by incubation with different dyes according to the manufacturer’s protocol. Then, supernatants were discarded, and cells were washed twice using pre-warmed DPBS. Finally, the images of stained cells were taken on Nikon A1 PLUS. The dyes information was listed as following: BODIPY (lipid detection, 3 μM, MCE, Cat. #HY-W090090); Nile Red (lipid detection, 0.5 μM, MCE, Cat. #HY-D0718); SPiDER-βGal (senescence detection, Dojindo, Cat. #SG02).

Immunofluorescence
For cells, MOVAS and RCA-SMCs were seeded on confocal dishes at an appropriate density. After treatments, the cells were washed with DPBS twice and then fixed with 4% formaldehyde buffer (PFA) at 37°C for 20 min. After washing 2-3 times with PBS, the cells were permeabilized and blocked with 5% BSA and 15% goat serum in 1× PBS for 1 h at 25°C. Then, the primary antibodies were incubated at 4°C overnight, and Alexa Fluor–conjugated secondary antibodies were incubated at 25°C for 2 h. DAPI was used to stain nuclei. All the graphs were reviewed and taken by Nikon A1 PLUS. All the used antibodies were summarized as following: SREBP1 (Proteintech, Cat. #66875-1-Ig, 1:200); SREBP2 (Proteintech, Cat. #28212-1-AP, 1:250); α-SMA (Proteintech, Cat. #14395-1-AP, 1:1000); Pan-Acetylation (Proteintech, Cat. #66289-1-Ig, 1:100); CD31 (Servicebio, Cat. #GB113151-100, 1:5000); Ki-67 (abcam, Cat. #ab16667, 1:2000); P21 (Servicebio, Cat. #GB115313-100, 1:200); SRF (Proteintech, Cat. #16821-1-AP, 1:200); MYOCD (Affinity Biosciences, Cat. #DF2434, 1:200); H3K9Ac (ABclonal, Cat. #A7255, 1:200); H3K27Ac (Beyotime, Cat. #AF5620, 1:200); IgG-mouse (Proteintech, Cat. #B900620, 1:500); IgG-rabbit (Proteintech, Cat. #30000-0-AP, 1:500).

Flow cytometry analysis
After specific treatment, 2×105 cells were collected and stained with different dyes according to the manufacturer’s protocol, followed by three washes with specific buffer and centrifugation at 500×g for 5 min at 25°C. Then, supernatants were discarded, and cells were resuspended in 300~500 μL buffer. Finally, the labeled cells were analyzed using a CytoFLEX (Beckman Colter Life Sciences) and FlowJo v.10 software (BD Life Sciences). The dyes information was listed as following: BODIPY (lipid detection, 3 μM, MCE, Cat. #HY-W090090); SPiDER-βGal (senescence detection, Dojindo, Cat. #SG02). Gating strategies were summarized in Supplementary Information file.

Enzyme linked immunosorbent assay (ELISA)
The cells were collected into a 1.5 mL centrifuge tube. 5×105 cells were dissolved in 200 μL extraction solution. After sonication for 3 min on ice, the extraction solution was centrifuged at 13,000×g for 10 min to collect supernatant. The supernatant was placed on ice to be tested. Acetyl-CoA, fatty acid (FA), and triglyceride (TG) levels were measured using an Acetyl-CoA ELISA Kit (Sangon Biotech, Cat. #D751001), FA ELISA Kit (Meimian Industrial Co., Ltd., Cat. #MM-71779R2), and TG ELISA Kit (Meimian Industrial Co., Ltd., Cat. #MM-0610R2) following the manufacturer’s protocol, respectively. The concentration was normalized with the protein content of each sample (BCA Protein Assay Kit, BioVision).

Measurement of citrate levels
Citrate levels in cells and tissue were measured by Citrate Content Assay Kit (Sorlarbio, Cat. #BC2155) following by the recommendations of the manufacturer. The citrate concentration and standard curve were established by an enzyme reader (TECAN, Cat. #30086376, Switzerland). The citrate level was normalized with the protein content of each sample (BCA Protein Assay Kit, BioVision).

Protein isolation and western blot analysis
Nuclear and cytoplasmic proteins were extracted with a nuclear and cytoplasmic protein extraction kit (Yeasen, Cat. #20126ES50) according to the manufacturer’s instructions. Cells, tissue and isolated nucleus samples were washed by ice-cold PBS and lysed with RIPA lysis buffer (Beyotime, Cat. #P0013B) supplemented with phosphatase and protease inhibitors (Roche). Lysates were separated on 10–12.5% SDS-PAGE gels (Beyotime, Cat. #P0012AC), and then transferred to 0.22–0.45 μm polyvinylidene difluoride membranes (Merck Millipore) by Trans-Blot System (Bio-Rad Laboratories). Membranes were then blocked with 5% skimmed milk diluted in TBST for 1 h at 25°C, and then were incubated in diluted primary antibodies overnight at 4°C. Subsequently, the membranes were incubated with secondary antibodies conjugated with horseradish peroxidase (HRP) for 1 h at 25°C. Western HRP substrate (Millipore) and imaging system were employed to blot detection. The antibodies used in this study are as followed: β-Tubulin (Abways, Cat. #AB0057, 1:2000); β-Actin (Sigma, Cat. #A5316, 1:10000); α-SMA (Proteintech, Cat. #14395-1-AP, 1:1000); SM22α (Proteintech, Cat. #, 1:1000); P21 (Servicebio, Cat. #GB115313-100, 1:500); SREBP1 (Proteintech, Cat. #66875-1-Ig, 1:1000); SREBP2 (Proteintech, Cat. #28212-1-AP, 1:1000); MYOCD (Affinity Biosciences, Cat. #DF2434, 1:1000); Pan-Acetylation (Proteintech, Cat. #66289-1-Ig, 1:500); ACLY (Proteintech, Cat. #67166-1-Ig, 1:1000); ACSS2 (Proteintech, Cat. #16087-1-AP, 1:1000); H3K27Ac (Beyotime, Cat. #AF5620, 1:1000); H3K9Ac (ABclonal, Cat. #A7255, 1:1000); H3K18Ac (PTMBIO, Cat. #PTM-114RM, 1:1000); H3 (Proteintech, Cat. #17168-1-AP, 1:1000); SRF (Proteintech, Cat. #16821-1-AP, 1:1000); LaminA/C (Proteintech, Cat. #10298-1-AP, 1:1000); p-AKT (abcam, Cat. #ab38449, 1:1000); AKT (abcam, Cat. #ab8805, 1:1000); mTOR (abcam, Cat. #ab134903, 1:1000); p-mTOR (abcam, Cat. #ab109268, 1:1000).

RNA extraction and real-time quantitative polymerized chain reaction (RT-qPCR)
Total RNA was extracted from carotid artery tissues and cells according to the protocols of Axypre™ Multisource Total RNA Miniprep kit (Axygen). The isolated RNA was then reverse-transcribed by using PrimeScript™ RT Mix with gDNA eraser (Takara). SYBR® Premix Ex Taq™ Ⅱ (Takara) was used for qRT-PCR by using 7500 system (Applied Biosystems). All the primers are listed in Supplementary Table 1. The relative mRNA expression was analysis using the comparative threshold cycle method (2−ΔΔCt) using Actb as the endogenous control gene.

Immunohistochemistry
After deparaffinization, serial sections (4 μm) were soaked in citric acid (pH=6.0) antigen retrieval buffer (Servicebio, Cat. #G1203) and heated twice for 10 minutes for antigen retrieval in a microwave oven. Then, the sections were placed in 3% hydrogen peroxide solution and incubated at 25°C in darkness for 25 min to block endogenous peroxidase activity. The sections were then incubated with primary antibodies. HRP-labeled antibodies (Servicebio, Cat. #GB23303, 1:200) were used as secondary antibodies. Nuclei were were counterstained using hematoxylin (Servicebio), and diaminobenzidine (Servicebio, Cat. #G1211) was served as the chromogen. The primary antibodies used in this study are as followed: α-SMA (Proteintech, Cat. #14395-1-AP, 1:100); P21 (Servicebio, Cat. #GB115313-100, 1:50); SREBP1 (Proteintech, Cat. #66875-1-Ig, 1:100); SREBP2 (Proteintech, Cat. #28212-1-AP, 1:100); IgG-mouse (Proteintech, Cat. #B900620, 1:500); IgG-rabbit (Proteintech, Cat. #30000-0-AP, 1:500).

RNA-seq analysis
Total RNA was extracted using Axypre™ Multisource Total RNA Miniprep kit (Axygen) according to the manufacturer’s protocol82. RNA quality was evaluated by Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and verified using RNase-free agarose gel electrophoresis. After treatment with Oligo(dT) beads, the enriched mRNA was then fragmented and reverse transcribed into cDNA using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB #7530, New England Biolabs, Ipswich, MA, USA). The double-stranded cDNA products were then purified by QiaQuick PCR extraction kit (Qiagen, Venlo, The Netherlands), end repaired, added poly(A), and ligated with Illumina sequencing adapters. The adapter-ligated products were size selected by agarose gel electrophoresis. Finally, PCR amplification and sequencing were completed using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. (Guangzhou, China). Raw reads were further filtered by fastp (V.18.0) to get high-quality clean reads. Short reads alignment tool Bowtie2 (V.2.2.8) was used for mapping reads to ribosome RNA (rRNA) database. The rRNA mapped reads then will be removed. The remaining clean reads were further used in assembly and gene abundance calculation. An index of the reference genome was built, and paired-end clean reads were mapped to the reference genome (Ensembl_release 110 for MOVAS; Ensembl release 109 for rats) using HISAT V.2.2.4 and other parameters set as a default. The mapped reads of each sample were assembled by using StringTie V.1.3.1 in a reference-based approach. For each transcription region, an FPKM (fragment per kilobase of transcript per million mapped reads) value was calculated to quantify its expression abundance and variations, using RSEM software. RNAs differential expression analysis was performed by DESeq2 software between two different groups. The genes with a false-discovery rate (FDR) < 0.05 and fold-change ≥ 1.5 or ≤ 1/1.5 were considered differentially expressed genes. KEGG, GO, and GSEA enrichment analysis was performed using the KEGG, Gene Ontology database (http://www.geneontology.org/), and GSEA database (https://www.gsea-msigdb.org/gsea/index.jsp). Regulatory target gene sets from the GSEA database were used for transcription factors enrichment analysis (https://www.gsea-msigdb.org/gsea/msigdb/index.jsp) as previously described83. All the bioinformatic analysis was performed by Gene Denovo Biotechnology Co., Ltd (Guangzhou, China).

Lipid extraction and lipidomics analysis
Lipidomics analysis was performed by Shanghai Applied Protein Technology Co., Ltd. (Shanghai, China)84. Briefly, samples were first mixed with 200 μL methanol, and then 10 μL internal lipid standards (SPLASH™ LIPIDOMIX™ Mass Spec Standard, AVANTI, 330707-1EA) and 800 μL methyl tert-butyl ether were added. The quality control (QC) samples are a composite prepared by pooling equal volumes of all the samples to be tested. The mixture was adequately vortexed, sonicated for 20 min at 4°C and then kept for 30 min at room temperature. After that, 200 μL of MS-grade water was added, and the mixture was vortexed and centrifuged at 20000×g for 15 min at 4°C. The upper organic solvent layer was obtained and dried under nitrogen. For LC-MS analysis, the samples were re-dissolved in 200 μL of IPA/CAN (9:1, v/v) solvent and centrifuged at 20000g at 4°C for 15 min, then the supernatant was injected. The analysis was performed on a UHPLC system (LC-30A, Shimadzu) coupled with QTRAP MS (6500+, Sciex). The analytes were separated on HILIC (Phenomenex, Luna NH2, 2.0 mm×100 mm, 3 μm) and C18 column (Phenomenex, Kinetex C18, 2.1×100 mm, 2.6 μm). For C18 separation, the column temperature was set at 45°C. Mobile phase A: 70% acetonitrile + 30% H2O + 5 mM ammonium acetate, mobile phase B: IPA solution. A gradient (20% B at 0 min, 60% B at 5 min, 100% B at 13 min, 20% B at 13.1–17 min) was then initiated at a flow rate of 0.35 mL/min. The sample was placed at 10°C during the whole analysis process. For Amino separation, the column temperature was set at 40°C. Mobile phase A: 2 mM ammonium acetate + 50% methanol + 50% acetonitrile, mobile phase B: 2 mM ammonium acetate + 50% acetonitrile + 50% H2O. A gradient (3% B at 0~3 min, from 3% to 100% B at 3~13 min, 100% B at 13~17 min, 3% B at 17.1~22 min) was then initiated at a flow rate of 400 μL/min. 6500+ QTRAP (AB SCIEX) was performed in positive and negative switch mode. The ESI positive source conditions were as follows: Source temperature: 400°C; Ion Source Gas 1 (GS1): 50 Ion Source Gas 2 (GS2): 55; Curtain Gas (CUR): 35; Ion Spray Voltage (IS): +3000 V; The ESI negative source conditions were as follows: Source temperature: 400°C; Ion Source Gas 1 (GS1): 50 Ion Source Gas 2 (GS2): 55; Curtain gas (CUR): 35; Ion Spray Voltage (IS): −2500 V. Multiple Reaction Monitoring method was used for mass spectrometry quantitative data acquisition, and Sciex OS was used for quantitative data processing by Shanghai Applied Protein Technology Co., Ltd. (Shanghai, China). The concentrations of lipids (ng/g, normalized to sample weight) were summarized in Supplementary Data 1. Following lipids were quantified: glycerophospholipids (including phosphatidic acid PA, phosphatidylcholine PC, phosphatidylethanolamine PE, phosphatidylglycerol PG, phosphatidylinositol PI, phosphatidylserine PS, lysophosphatidic acid LPA, lysophosphatidylethanolamine LPE, lysophosphatidylserine LPS), Sphingolipids (including ceramide Cer, dihydroceramide DHCer, hexoceramide Hex1Cer, dihexose ceramide Hex2Cer, sphingomyelin SM), Glycerolipids (including monoglycerides MG, diglyceride DG, triglycerides TG), cholesteryl ester (ChE), and fatty acids (FA).

Immunoprecipitation and mass spectrometry (IP-MS)
For protein IP-MS, the cell lysates treated by Cell Lysis Buffer (CST, Cat. #9803S) were incubated with Pan-Acetylation (Proteintech, Cat. #66289-1-Ig) or IgG (Proteintech, Cat. #B900620) primary antibody overnight at 4°C. Immunoprecipitates were collected and separated by SDS–PAGE. The gel lysis and protein digestion were performed by the commercially available iST Sample Preparation kit (PreOmics, Germany). The peptides were analyzed by nano-HPLC–MS/MS, and the resulting data were processed by PEAKS Studio version 10.6 (Bioinformatics Solutions Inc., Waterloo, Canada) by Gene Denovo Biotechnology Co. (Guangzhou, China). The results were summarized in Supplementary Data 2 and 3.

ATAC-seq library preparation and sequencing
Briefly, cells were transferred into 5 μL of lysis buffer (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, and 0.005% digitonin) in ice for 10 min. The ATAC reaction was performed by adding 4 μL of ddH2O, 4 μL of TTBL (5×), and 5 μL TTE mix V5 (Vazyme Biotech, TD711) at 37°C for 30 min and then stopped by adding 5 μL of TS (5×) stop buffer at 25°C for 5 min. DNA was extracted using ATAC DNA Extract Beads. Then, DNA was dissolved in 20 μl ddH2O and PCR-amplified with 10 μL of index primers (Vazyme Biotech, TD202), 30 μL of CAM (2×) (Vazyme Biotech, TD711) with the program of 72°C for 3 min; 95°C for 3 min; 98°C for 10 s, 60°C for 5 s for 12 cycles; and 72°C for 1 min. The amplified DNA was size-selected using ATAC DNA Clean Beads (Vazyme Biotech, TD711). The libraries were multiplexed and then sequenced using an Illumina novaseq 6000 with PE150 method by IGENEBOOK Biotechnology Co., Ltd (Wuhan, China, http://www.igenebook.com). The bioinformatic analysis was performed by Vazyme Biotech Co., Ltd (Nanjing, China).

Statistics and reproducibility
Data were expressed as mean±SD. Statistical analysis and graphical representation were performed in the GraphPad Prism software (v. 9.0.0) or Rstudio (version 4.1). Normality of the data was evaluated using the Shapiro-Wilk test. Two-tailed unpaired Student t tests were used to assess the differences between two groups, when normal distribution was satisfied; otherwise, the Mann–Whitney test was used. The differences between multiple groups were compared using one-way ANOVA or two-way ANOVA, followed by Dunnett’s (one-way) or Tukey’s (two-way) multiple comparisons test, when normal distribution was satisfied; otherwise, the nonparametric Kruskal–Wallis test was performed for further analysis. Two-way mixed-effects ANOVA followed by Sidak’s multiple comparisons test was used to analyze differences in body weight at multiple time-points in order to account for the correlation of repeated measurements of the same rats (Supplementary Fig. S1B; S13H). Statistical significance was set at P < 0.05 (*); two asterisks (**) for P < 0.01; three asterisks (***) for P < 0.001; and four asterisks (****) for P < 0.0001. The representative data shown as pathological staining, immunohistochemistry, immunofluorescence, WB, TEM images were obtained from at least three independent experiments (for Figs. 1B, G, H, J, K; 2C, D; 3C–E, G, H; 4F, K, M, P; 5C, D, H, K, M; 6J; 7F, N).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study by Bowen Li et al. investigated the role of SREBP1 governed lipogenesis in microgravity-induced senescence of vascular smooth muscle cells. Mechanistically, SREBP1-mediated lipogenesis upregulates acetyl-CoA pool, thus regulates chromatin accessibility and inhibits SRF/myocardin activity. This study revealed a novel role of SREBP1 in SMC identity and vascular senescence. The manuscript is well-written. However, the lack of several key evidence makes this work not that convincing. The authors need to appropriately address all comments raised below.\nMajor concerns\n1. Novelty. Senescence of vascular cells has been confirmed as the core event of vasculature aging which plays a key role in morbidity and mortality of people. In the past 20 years, plenty of studies revealed that genomic instability, chronic low-grade inflammation, oxidative stress, deregulated nutrient sensing etc. contribute to vascular senescence (Circulation. 2003;107:139–146; Cell. 2013;153:1194–1217; Cardiovasc Res. 2018;114:513–528.). This study, for the first time, hypothesized that activation of sterol regulatory element-binding protein 1 (SREBP1) and lipogenesis promotes vascular senescence. The finding, if is supported by solid evidence, may add a piece to the puzzle in the research of vascular senescence. However, the data provided by the authors are weak. The following suggestions may help.\n1) The authors should perform more, some are key, experiments to identify senescence both in vitro and in vivo, such as β-gal staining, SASP etc., according to the published papers (Circ Res. 2021 Feb 19;128(4):474-491; Eur Heart J. 2024 Aug 1: ehae379.).\n2) SMC specific SREBP1 knockout mice underwent simulated microgravity model should be used to clarify the role of SMC-SREBP1 in vivo. Note that pharmacological inhibition or virus lacks tissue specificity. Additionally, is endothelial cell SREBP1 involved in vasculature aging under microgravity?\n3) The authors provided evidence that pharmacological activation of SREBP1 promotes senescence. However, this makes the phenotype of SREBP1 appear artificial. The activator of SREBP1 in microgravity condition should be investigated. Are there any gravity sensors (cytoskeleton or others) or signaling sensing microgravity contribute to SREBP1 activation? Do classical senescence inducers such as H2O2 activates SREBP1? Knowing this will make the story a whole picture.\n4) More evidence should be provided to make the SREBP1-lipogensis-acetyl-CoA-SRF/myocardin axis more convincing in this model. Could depletion of acetyl-CoA transporter or key enzymes in acetyl-CoA biogenesis pathway block the effect of SREBP1 activation? Can supplementation of acetyl-CoA mimics the phenotype of SREBP1? How hyper-acetylation of chromatin affects expression of aging genes in this model? Which histone acetyltransferase is involved in this process? In addition, could SREBP1 transcriptionally regulate aging genes?\n5) The choosing of SREBP1 may be artificial according to the bulk RNA-seq data. The most significant pathway enriched is steroid biosynthesis, why the role of steroid biosynthesis and SREBP2 are neglected, note that SREBP2 is also activated in HU or SM?\n2. Experiments design.\n1) The phenotype of SREBP1 should be also investigated in human VSMCs.\n2) Missing controls- Fig3 (IgG), Fig4L (0 h), Fig4P (positive control such as H2O2) etc.\n3) Repeat the in vitro assays to make the N is at least 6.\n3. Graphs. Most of the figures are of low resolution and poor quality, which should be replaced in the next version.\nMinor concerns\n1. In Figure1 F, \"P21\" is marked as \"P2\".\n2. In Figure7 B, \"B-tubulin\" needs to be in the same format as above.\n3. The analysis of sequence data (Fig2, 4, 6) should be moved to supplemental materials.", "reviewer_2": "The manuscript describes a novel potential role of SREBP1 in dedifferentiation and senescence of vascular smooth muscle cells through a mechanisms of epigenetic remodeling.\nThe rationale of the original study was to assess the mechanisms of vascular remodelling following changes in microgravity.\n- Reports have been published about the potential role and mechanistic pathways involving SREBP1 in atherogenesis and what effects gene expression has on EC and SMC function. However, the effect of SREBP1 in microgravity induced SMC senescence has not been described before.\n- My major comment concerns the models for studying microgravity. This is not the major expertise of this reviewer but as an outsider I am surprised that this rat model is accepted as a standard to study effects of microgravity on (for example) muscle dystrophy. The same accounts for the 2-dimensional cell culture models. The animal is still under the influence of gravity (just like the cells). The animal is captured with his hindlimb in a cage which will likely also affect general metabolism, feeding patterns and subsequently maybe insulin levels (that control SREBP1). But questioning the models also questions the relevance of the outcomes for vascular pathology.\n- The effects of gravity on organ systems is a scientific exercise of interest but the inferences for vascular disease may be limited. Also experiments have been executed to search for the translation to effects on cell metabolism in SMCs that are not affected by gravity. (For example in Figure 7). The line of thought is therefore a bit difficult to follow. The manuscript starts with differential expression studies using gravity models and it seems that in the final experiments (the regular SMC culture, non-MOVAT) confirmation is found in non MOVAT cultures while these were in fact controls in the differential expression studies.\n- If the authors suggest that SREBP1 does have an important function in cell plasticity not just under the influence of microgravity but also in general vascular pathology, then an attempt should be made to study the role of SREBP1 in SMC senescence either in a pathological animal model (such as atherosclerosis) or search for confirmation of pathways in human pathological samples (such as single cell seq of atherosclerotic specimen, data are available online).", "reviewer_3": "Simulated microgravity (SM) induces a senescence-like modification of carotid artery (CA) in both human and animal observations, but the underlying mechanism is not completely clear. In this manuscript, Li and colleagues reported that SM activated SREBP1-mediated lipogenesis and upregulated acetyl-CoA pool to increase histone acetylation and alter the chromatin accessibility. As a consequence, the transcription of contractile genes was reduced and the transcription of aging genes was induced, which results in a senescence-like remodeling of carotid artery. Overall, this study is interesting.\nHowever, some conclusions are not strongly supported by the data presented and some data quality needs to be improved.\nMajor concerns:\n1. In adipocytes, SREBP1c protects against adipocytes by regulating PARP1 activity (Lee et al, 2022). Muscle PARP1 inhibition can extend the lifespan of Drosophila (Guo et al, 2023). Does SREBPs-PARP1 play a role in SM and is it possible that SM promotes VSMCs senescence by activating this pathway?\n2. Activation of SREBP1 can induce increased pan-ac (including histone acetylation). But why some genes were up-regulated (aging genes) and others were down-regulated (contractile gens)? What causes selective gene regulation and different effects on chromatin accessibility? Moreover, as LXR-623 induced acetylation of addition 34 proteins, why authors only focused on histone acetylation? Based on the model presented in Figure 8, the authors assumed that the induction of histone acetylation is the main cause for elevated expression of aging genes. But there is no data showing it is acetylation of histones but not other proteins. For Figure 5: This study should be performed under SM condition.\n3. Several conclusions in this manuscript are not well supported by the data. Many data show only correlation but no causal relationship can be derived. For example, Figure 6J showed reduced nuclear translocation of SRF and MYOCD by LXR-623. But this result cannot support the conclusion that “The reshaped chromatin accessibility further decreased the nuclear translocation of SRF and MYOCD (a transcriptional coordinator of SRF), which partially contributed to transcriptional inactivity of SRF (Figure 6J).”\nFigure 5: There is no evidence showing “Upregulated Ac-CoA pool is pivotal in the SREBP1-activation triggered senescence-like transformation of VSMCs.” The data presented are correlation between increased Ac-CoA, protein acetylation, and senescence phenotype. No causal relationship can be obtained from current data. Since ACSS2 and ACLY-mediated acetyl CoA plays a pivotal role in VSMCs senescence induced by SREBP1 activation, what is the effect of knockdown of ACSS2 and ACLY in SM-treated VSMCs? Can addition of Ac-CoA (NaAc) rescue the phenotype caused by SREBP1 knockdown?\n4. Figure 7H-O: It is good to see authors examined the role of SREBP1 in SM-induced senescence phenotype in vivo. However, it is recommended to examine additional factors, including P21 staining, β gal staining, the transcription of contractile genes and aging genes.\n5. Figure S2E-S2F: Figure S2E show that SM inhibits the proliferation of MOVAS. Figure S2F show that SM killed MOVAS cells. The authors should show the detail of experiments. Whether the SM induced cell senescence or killed young cells? How to exclude the role of apoptosis on transformation phenotype and functional decline of vascular?\n6. It has been established that Ac-CoA is compartmentalized inside cells, including cytoplasm, mitochondria and nucleus. Histone acetylation increases could be more associated with nucleus acetyl-CoA. I am curies what pools of acetyl-CoA were significantly enhanced in MOVAS after both LXR-623 and SM treatments?\nMinor concerns:\n1. The quantification of Western blots is inaccurate. For example, Figure 3F (n-SREBP1), 5J (SMA), 5L (p21), 6J (MYOCD, no band but have grayscale) value, etc. The authors should also mark the non-specific bands in figure 3C, 4F, M.\n2. Page 5, line 16, line 19: should be Figure 1B-C, Figure 1D-F.\n3. The role of acetyl-CoA and ACSS2 in histone acetylation and senescence has been reported (PMID: 33917812; 34183849). The authors should discuss this point in their work.\n4. The key materials and methods used in this study should be included in the main text instead of putting in the supplementary materials.\n5. There is no description of how authors performed analysis for GSEA based activated TF enrichment analysis.\n6. Fig. 1F: p2 should be p21.", "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": "The manuscript titled “SREBP1-mediated lipogenesis promotes dedifferentiation and senescence of vascular smooth muscle cells through epigenetic remodeling” by Li et al. aims to mechanistically explain the phenomenon of VSMCs senescence and senescence-like modifications in carotid artery upon simulated microgravity. Authors use in vitro, ex vivo and in vivo models together with a range of molecular biology, biochemistry, imaging and omics techniques to demonstrate that VSMC senescence occurs through cellular epigenetic remodeling caused by SREBP1-mediated lipogenesis.\nAdvances in space exploration justify this study and the overall need to better understand the detrimental effect of microgravity on human body. It is evident that Authors have substantial experience in utilizing the technique of simulated microgravity. I appreciate the amount of work that has been done by Authors.\nEven though this complex study provides substantial amount of data, presented results do not support the entireness of tested hypothesis. The Authors provide data that demonstrates the involvement of SREBP1 in senescence-like processes in carotid arteries, however, evidence that lipogenesis promotes senescence via epigenetic remodeling is limited. Also, the manuscript must go through an extensive proofreading process first as the text for both the main and supplementary part has a lot of typos and mistakes (page 3 line 14 tunica media vasorumta, Fig1L ‘cell differention’, etc).\nMajor issues:\n1. Authors do not provide enough details for some of the assays essential for hypothesis testing. The major concern is around “Absolute Quantitative Lipidomics”. Authors do not provide any information or references around chromatographic columns they used, solvent gradients for lipid separation, list of internal standards and their quantities, mass spectrometry parameters, and overall, a method that was used for absolute lipid quantification making this part of the study non-reproducible. If the targeted lipid analysis was used, the authors must describe how absolute lipid quantities were estimated. How did Authors confirm (Table S2) the fatty acid composition including the exact fatty acid sn-position of PC lipids? How abundances of isomeric lipid species were estimated (e.g., TG46:1) while these molecules coelute as one peak (RT 8.61 min), however, Authors managed to quantify individual isomers? Are the numbers in Table S2 expressed as absolute concentrations? If so, what are the values (nM, ng)? I assume that these values were normalized to some amount of material taken for the analysis. If so, what is it – mg pf protein or wet sample weight? If absolute quantitative lipid analysis was performed, what was the utility of lipid ELISA assay, especially for TG and FA. How comparable is the data from the mass spectrometry vs ELISA?\n2. Cell proliferation cannot be estimated using cell viability assay. Why didn’t authors use the gold standard Ki-67 as the proliferation marker?\n3. For the 2D SM cell models, it is not clear what the control was. Supplementary page 4 line 20 – “Cells without rotation (1 g) were cultured in the same manner as the control group.” If one guided by corresponding Figure S2A, cultured cells in control group (CTRL) are covered with small layer of culture medium allowing cells to grow under normoxic conditions, while in case of SM cell model, the bottle is filled with media that will force cells to be under more hypoxic conditions as well as experience increased pressure due to the amount of liquid on top of them. If this is the case, observed lipid droplet accumulation and cell viability and senescence could be attributed to the experimental setup.\n4. Authors claim that SM in rat carotid artery (CA – confusing acronym as CA is used for citric acid in the manuscript) using HU model causes senescence-like remodeling in VSMCs by showing an increase in relative mRNA levels of genes associated with senescence (Fig 1). Carotid artery contains other cell types (endothelial cells, fibroblasts, dendritic cells, etc). There is no data presented showing that the signal increase is coming specifically from VSMCs alone.\n5. My understanding that the heatmap (Fig 2F) is derived from the targeted lipid analysis data in Table S2. If so, why do authors use just the data from only 3 HU rats, while the dataset in Table S2 has lipid data of 4 HU rats. What was the reason to omit one of the replicates? Same for Fig S5C (PCA analysis). And what was used as QC – not clear.\n6. Quality of Western Blots. Fig 4F – loading control for n and p-SREBP2 is saturated, too much material was loaded. Same for Fig 5G, Fig S13D – I am not sure if quantification is correct. Poor WB quality – Fig Fig S11I, Fig S13C.\n7. Given the fact that SM has profoundly different effect on fatty acid synthesis (Fig 2B) between the rat HU model and MOVAS SM model (e.g. Fasn difference 1.9 fold change increase in rat model vs 0.86 in MOVAS SM, Acly – 2.5 vs 0.67), what was the rational to move away from the HU rat model and test the hypothesis on MOVAS SM cell model? It is easy to test the drug in tissue culture, however, what grounds do Authors have to claim that similar mechanisms occur in vivo?\nOther comments:\n1. P 5 L16 – missing Fig number.\n2. P 5 L 15-16 – quantification of lumen area is not present. Authors claim that the lumen area is unchanged, however, there is no difference in the cross-section diameter of RCCA, although cIMT in HU is significantly increased. Please comment.\n3. P5 L20 and Fig 1F – there is no corresponding image of SMA staining that can confirm that the p21 signal comes from SMCs\n4. No data on mRNA expression of Ascc1 or Dgat1 was presented. ASCC1 is mitochondrial protein and can contribute to Acetyl-CoA pool, DGAT1 can play a compensatory role if DGAT2 is absent or inhibited. It is surprising to see an accumulation of lipid droplets filled with TG (based on your lipidomics data), however, Dgat2 expression is downregulated in HU or SM (Fig 2B). How would you explain higher TG biosynthesis without corresponding upregulation of Dgats? Decreased TG lipolysis? Need data showing this if so…\n5. Fig 1K – difficult to appreciate the difference – please provide quantification for this assay.\n6. P 6 L 29-32 – claimed increase in FA isn’t supported by data on Fig S5C-E (green dot for FA indicates almost no change).\n7. P 6 L 32-33 – reference to the Figure S6 does not correspond to the actual Figure S6."}, "author_response": "We sincerely appreciate the reviewer's insightful and constructive comments, which have significantly strengthened our manuscript. In this revised version, we have carefully addressed all the concerns raised by the reviewer and incorporated additional experiments, data analyses, and textual revisions to provide further mechanistic support for our conclusions. Below, we provide point-by-point responses to each comment.\nMajor concerns\n1. Novelty. Senescence of vascular cells has been confirmed as the core event of vasculature aging which plays a key role in morbidity and mortality of people. In the past 20 years, plenty of studies revealed that genomic instability, chronic low-grade inflammation, oxidative stress, deregulated nutrient sensing etc. contribute to vascular senescence (Circulation. 2003;107:139–146; Cell. 2013;153:1194–1217; Cardiovasc Res. 2018;114:513–528.). This study, for the first time, hypothesized that activation of sterol regulatory element-binding protein 1 (SREBP1) and lipogenesis promotes vascular senescence. The finding, if is supported by solid evidence, may add a piece to the puzzle in the research of vascular senescence. However, the data provided by the authors are weak. The following suggestions may help.\n1) The authors should perform more, some are key, experiments to identify senescence both in vitro and in vivo, such as β-gal staining, SASP etc., according to the published papers (Circ Res. 2021 Feb 19;128(4):474-491; Eur Heart J. 2024 Aug 1: ehae379.).\nResponse:\nThank you for the valuable suggestion. As recommended, we have performed the following experiments to identify senescence both in vitro and in vivo:\n1) β-gal staining in both MOVAS cells (in vitro) and HU model (in vivo) (revised Figure 1F, K), showing increased activity of β-gal after simulated microgravity.\n2) Expression of key SASP genes (e.g., Il6, Ccl2) via RT-qPCR (revised Figure 1E, I), showing increased expression of SASP genes in the SM or HU groups vs. controls.\nThese results, consistent with prior studies 1,2, further validate the senescence phenotype in our model. The Results section has been updated (page 5, lines 13-19).\n<revised Figure 1F, K>\n➢ Fig. 1F. Representative staining of β-Gal in the carotid artery from CON and HU rats. Scale bar = 2 mm.\n➢ Fig. 1K. Representative images of SPiDER-βGal staining (upper panel) or senescence β-Galactosidase staining (lower panel) of MOVAS after CTRL or SM treatment. Scale bar=50 μm.\n<revised Figure 1E, I, new>\n➢ Fig 1E. Relative expression levels of VSMC contractile genes, aging genes, and SASP genes in carotid artery from CON and HU rats. (n=3; *P<0.05, **P<0.01, ***P<0.001)\n➢ Fig 1I. Relative expression levels of VSMC contractile genes, aging genes, and SASP genes in MOVAS after control (CTRL) or stimulated microgravity (SM) treatments. (n=6; **P<0.01, ***P<0.001; ****P<0.0001)\n2) SMC specific SREBP1 knockout mice underwent simulated microgravity model should be used to clarify the role of SMC-SREBP1 in vivo. Note that pharmacological inhibition or virus lacks tissue specificity. Additionally, is endothelial cell SREBP1 involved in vasculature aging under microgravity?\nResponse:\nWe thank the reviewer for highlighting the need for VSMC-specific genetic models. To specifically investigate the role of VSMC-SREBP1 in vivo, we performed VSMC-specific SREBP1 knockdown using local delivery AAV-Tagln-sh-Srebf1 via Laponite hydrogels to the mouse carotid artery. This approach achieved:\n1) High VSMC specificity:\nLaponite hydrogels mixed with AAV-Tagln-mCherry effectively introduced the target sequences into VSMC (α-SMA+) of the mice carotid artery (revised Figure S19C).\n2) Phenotypic confirmation of VSMC-specific Srebf1 knockdown:\n⚫ Attenuated HU-induced media derangement and elastin break (revised Figure S19D).\n⚫ Mitigated cIMT thickening without altering lumen diameter (revised Figure S19E-H).\n⚫ Recovered the expression of SREBP1-related, aging, and contractile genes (revised Figure S19I-L).\nThese VSMC-specific results confirm our initial findings and strongly support the crucial role of VSMC-SREBP1 in microgravity-induced vascular remodeling.\n<revised Figure S19A-L, new>\nFigure S19. Establishment and evaluation of an in situ VSMC specific Srebf1 knockdown mice by AAV\n(A) Schematic diagram of rescue study of HU mice model.\n(B) The adventitia delivery of the Laponite hydrogels to the common carotid arteries of the mice.\n(C) Representative confocal microscopy images of mCherry (red) and α-SMA (green) of the common carotid arteries treated with AAV encoding mCherry loaded in the Laponite hydrogels 28 days after the gel injection. Scale bar = 50 μm.\n(D) Representative Verhoeff's Van Gieson staining (EVG) in the carotid artery of mice treated with AAV-Tagln-sh-Ctrl+CON, AAV-Tagln-sh-Ctrl+HU, AAV-Tagln-sh-Srebf1+CON, and AAV-Tagln-Srebf1+HU. Broken elastic fibers are indicated by red arrows in EVG staining. “L” indicates the side of vascular lumen. Scale bar = 12.5 μm.\n(E) Representative ultrasound images in the carotid artery of mice treated with AAV-Tagln-sh-Ctrl+CON, AAV-Tagln-sh-Ctrl+HU, AAV-Tagln-sh-Srebf1+CON, and AAV-Tagln-Srebf1+HU.\n(F) Quantifications of carotid intima-media thickness (cIMT) of right common carotid artery of rats treated with AAV-Tagln-sh-Ctrl+CON, AAV-Tagln-sh-Ctrl+HU, AAV-Tagln-sh-Srebf1+CON, and AAV-Tagln-Srebf1+HU. (n=6; **P<0.01, ***P<0.001)\n(G) Quantifications of end-diastolic lumen diameter of right common carotid artery (RCCA Diam; D) (mm) of mice treated with AAV-Tagln-sh-Ctrl+CON, AAV-Tagln-sh-Ctrl+HU, AAV-Tagln-sh-Srebf1+CON, and AAV-Tagln-Srebf1+HU. (n=6)\n(H) Quantifications of end-systolic lumen diameter of right common carotid artery (RCCA Diam; S) (mm) of mice treated with AAV-Tagln-sh-Ctrl+CON, AAV-Tagln-sh-Ctrl+HU, AAV-Tagln-sh-Srebf1+CON, and AAV-Tagln-Srebf1+HU. (n=6)\n(I) Relative mRNA expression levels of Srebf1, Acly, Acss2, Acta2, Tagln, Cdkn1a, and Trp53 in CA of mice treated with AAV-Tagln-sh-Ctrl+CON, AAV-Tagln-sh-Ctrl+HU, AAV-Tagln-sh-Srebf1+CON, and AAV-Tagln-Srebf1+HU. (n=6; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001)\n(J) Representative immunofluorescence images of IgG (mouse, red), IgG (rabbit, green), and IgG (647, cyan) in the carotid artery from CON and HU mice. Scale bar = 100 μm.\n(K) Representative immunofluorescence images of SREBP1 (red), α-SMA (green), and CD31 (cyan) in CA of mice treated with AAV-Tagln-sh-Ctrl+CON, AAV-Tagln-sh-Ctrl+HU, AAV-Tagln-sh-Srebf1+CON, and AAV-Tagln-Srebf1+HU. Scale bar = 20 μm.\n(L) Representative immunofluorescence images of P21 (red), α-SMA (green), and CD31 (cyan) in CA of mice treated with AAV-Tagln-sh-Ctrl+CON, AAV-Tagln-sh-Ctrl+HU, AAV-Tagln-sh-Srebf1+CON, and AAV-Tagln-Srebf1+HU. Scale bar = 20 μm.\nMoreover, our following in vivo and in vitro data demonstrated that endothelial cells (EC)-SREBP1 does not significantly contribute to vasculature aging under microgravity:\n1) In vivo evidence (revised Figure 1H, 3D):\nImmunofluorescence analysis revealed that HU-induced upregulation of P21 and SREBP1 was predominantly localized to VSMC (α-SMA+) but not EC (CD31+) in the carotid artery.\n2) In vitro evidence (revised Figure S2G-I, S7G):\nHUVEC (a canonical model of EC) exposed to simulated microgravity showed no activation of SREBPs and no significant senescence phenotypes, including increased cell size, upregulation of aging or SASP markers.\nAll these findings suggest that vascular aging under microgravity is primarily mediated by VSMC, with minimal EC involvement. The Results section has been updated (page 13, line 26 to page 14, line 2).\n<revised Figure 1H, 3D, new>\n➢ Fig. 1H. Representative immunofluorescence images of P21 (red), CD31 (cyan), and α-SMA (green) in the carotid artery from CON and HU rats. Intensity traces (offset white lines) are plotted at right. “L” indicates the side of vascular lumen. Scale bar = 200 μm or 100 μm (zoom in panel).\n➢ Fig. 3D. Representative immunofluorescence images of SREBP1 (red), CD31 (cyan), and α-SMA (green) in the carotid artery from CON and HU rats. Intensity traces (offset white lines) are plotted at right. “L” indicates the side of vascular lumen. Scale bar = 200 μm or 100 μm (zoom in panel).\n<revised Figure S2G-I, S7G, new>\n➢ Fig. S2G. Representative FACS images of HUVEC after CTRL or SM treatment. FSC=forward scatter; SSC=side scatter.\n➢ Fig. S2H. Relative mRNA expression levels of CDKN1A, CDKN2A, TP53, IL6, and CCL2 of HUVEC after CTRL or SM treatment. (n=6)\n➢ Fig. S2I. Immunoblot analyses of P21 of HUVEC after CTRL or SM treatment. Band quantification is normalized to the first lane.\n➢ Fig. S7G. Relative mRNA expression levels of SREBF1, SREBF2, FASN and ACACA of HUVEC after CTRL or SM treatment.\n3) The authors provided evidence that pharmacological activation of SREBP1 promotes senescence. However, this makes the phenotype of SREBP1 appear artificial. The activator of SREBP1 in microgravity condition should be investigated. Are there any gravity sensors (cytoskeleton or others) or signaling sensing microgravity contribute to SREBP1 activation? Do classical senescence inducers such as H2O2 activates SREBP1? Knowing this will make the story a whole picture.\nResponse:\nWe appreciate the reviewer’s insightful suggestions to strengthen the physiological relevance of our findings. We have addressed these points as follows:\n1) Firstly, the pharmacological SREBP1 activation served as a controlled approach to investigate the direct relationship between SREBP1 activation and senescence-like transformation in VSMC, validating the contributing role of VSMC-SREBP1. Our pharmacological approach complements the physiological microgravity data (Figures 1-3) by specifically establishing SREBP1's causal role in senescence, consistent with standard mechanistic studies.\n2) Secondly, to investigate the activator of SREBP1 in microgravity condition, we focused on the mTOR pathway based on its established role in SREBP1-mediated lipogenesis, mechanotransduction, and senescence. 3-7 Our data demonstrate that simulated microgravity activates mTOR signaling by upregulating the ratio of p-mTOR/mTOR and p-AKT/AKT (Figure S7H-I), suggesting mTOR as a potential upstream regulator of SREBP1 in this process. Further studies are needed to fully elucidate this mechanism.\n3) Thirdly, based on the reviewer’s suggestion, we extended our investigation to classical senescence inducers and found that H O treatment induced: (a) cellular senescence, 2 2 as evidenced by higher β-galactosidase activity, enlarged cell size, and upregulated expression of senescence-associated genes (Trp53 and Cdkn1a); (b) intracellular lipid accumulation; (c) upregulation of SREBP1 and its target genes (Acaca and Fasn) (Figure S9), which is consistent with the previous findings. 8\nAll these findings suggest a pivotal role for SREBP1 in VSMC senescence induced by H O or simulated microgravity, and implicated that the mTOR signaling may function as a 2 2 sensor bridging the gap between VSMC gravity sensing and SREBP1 activation. The Results section has been updated (page 8, lines 5-9 and lines 29-31).\n<revised Figure S7H-I, new>\n➢ Fig. S7H. Immunoblot analyses of p-AKT, AKT, p-mTOR, and mTOR of MOVAS treated with CTRL or SM.\n➢ Fig. S7I. Immunoblot analyses of p-mTOR and mTOR of HASMC treated with CTRL or SM. Band quantification of p-mTOR/mTOR is normalized to the first lane.\n<revised Figure S9, new>\nFigure S9. Cell senescence inducer (H2O2) directly activates SREBP1-mediated lipogenesis\n(A) Representative FACS images of MOVAS after PBS or H2O2 (600 mM) treatment. FSC=forward scatter; SSC=side scatter.\n(B) Representative images of bright field (upper panel) or senescence β-Galactosidase staining (lower panel) of MOVAS after PBS or H2O2 (600 mM) treatment. Scale bar=200 μm.\n(C) Lipid level in MOVAS after PBS or H2O2 (600 mM) treatment by BODIPY staining (left panel) and the relative quantifications of BODIPY mean fluorescence intensity (MFI) (right panel). (n=6; **P<0.01)\n(D) Relative mRNA expression levels of Trp53, Cdkn1a, Srebf1, Acaca, and Fasn of MOVAS after PBS or H2O2 (600 mM) treatment. (n=6; ***P<0.001, ****P<0.0001)\n(E) Immunoblot analyses of P21 and SREBP1 of MOVAS treated PBS or H2O2 (600 mM). Band quantification is normalized to the first lane.\n(F) Lipid level in HASMC after PBS or H2O2 (600 mM) treatment by BODIPY staining (left panel) and the relative quantifications of BODIPY mean fluorescence intensity (MFI) (right panel). (n=6; ****P<0.0001)\n4) More evidence should be provided to make the SREBP1-lipogensis-acetyl-CoA-SRF/myocardin axis more convincing in this model. Could depletion of acetyl-CoA transporter or key enzymes in acetyl-CoA biogenesis pathway block the effect of SREBP1 activation? Can supplementation of acetyl-CoA mimics the phenotype of SREBP1? How hyper-acetylation of chromatin affects expression of aging genes in this model? Which histone acetyltransferase is involved in this process? In addition, could SREBP1 transcriptionally regulate aging genes?\nResponse:\nWe sincerely appreciate the reviewer's insightful suggestions. In response, we have conducted additional experiments to strengthen the mechanistic evidence supporting the SREBP1-lipogenesis-acetyl-CoA-SRF/myocardin axis in simulated microgravity-induced vascular senescence. Our key findings are summarized below:\n1) Inhibition of acetyl-CoA biogenesis block the effect of SREBP1 activation:\nKnockdown of Acly or Acss2 (two essential genes for acetyl-CoA biogenesis) significantly attenuated SM-induced SREBP1 activation, lipid accumulation, and cellular senescence (revised Figure S15F-L).\n<revised Figure S15F-L, new>\n(F) Relative mRNA expression levels of Acly of MOVAS transfected with different targeting siRNAs. (n=6; ****P<0.0001)\n(G) Immunoblot analysis of ACLY of MOVAS transfected with si-Acly #2. Band quantification is normalized to the first lane.\n(H) Relative mRNA expression levels of Acss2 of MOVAS transfected with different targeting siRNAs. (n=6; ****P<0.0001)\n(I) Immunoblot analysis of ACSS2 of MOVAS transfected with si-Acss2 #2. Band quantification is normalized to the first lane.\n(J) Lipid level in MOVAS treated with CTRL+siCtrl, SM+siCtrl, SM+si-Acly, and SM+si-Acss2 by BODIPY staining (left panel) and the relative quantifications of BODIPY mean fluorescence intensity (MFI) (right panel). (n=6; **P<0.01, ***P<0.001, ****P<0.0001)\n(K) Senescence level in MOVAS treated with CTRL+siCtrl, SM+siCtrl, SM+si-Acly, and SM+si-Acss2 by SPiDER-βGal staining (left panel) and the relative quantifications of SPiDER-βGal mean fluorescence intensity (MFI) (right panel). (n=6; **P<0.01, ***P<0.001, ****P<0.0001)\n(L) Immunoblot analyses of SREBP1 and P21 in MOVAS treated with CTRL+siCtrl, SM+siCtrl, CTRL+ si-Acly, SM+si-Acly, CTRL+ si-Acss2, and SM+si-Acss2. Band quantification is normalized to the first lane.\n2) Acetyl-CoA supplementation via sodium acetate (NaAc, acetyl-CoA precursor) mimics SREBP1 activation:\n• Increased intracellular acetyl-CoA and global protein acetylation (revised Figure S14A-B).\n• Recapitulated SREBP1 activation effects, including elevated H3 acetylation (H3K9Ac, H3K27Ac), reduced Acta2, and increased Cdkn1a expression (revised Figure S14A, 5J-K).\n• Partially rescued senescence phenotypes in SREBP1-knockdown cells (revised Figure S14H-J).\n< revised Figure S14A-B, 5J-K, modified>\n➢ Fig. S14A. Immunoblot analyses of Pan-Ac, H3K9Ac, and H3K27Ac in MOVAS treated with PBS, and NaAc (40 mM, 80mM, 120 mM, 160 mM, 200 Mm) for 48h.\n➢ Fig. S14B. Relative levels of Ac-CoA in MOVAS treated with DMSO + Vehicle, DMSO + NaAc (120 mM), LXR-623 + Vehicle, LXR-623 + NaAc (120 mM) (48h). (n=6; *P<0.05, **P<0.01, ****P<0.0001)\n➢ Fig. 5J. Relative mRNA expression levels of Acta2 and Cdkn1a in MOVAS treated with DMSO + Vehicle, DMSO + NaAc (120 mM), LXR-623 + Vehicle, LXR-623 + NaAc (120 mM) (48h). (n=6; ***P<0.001, ****P<0.0001)\n➢ Fig. 5K. Immunoblot analyses of α-SMA, SM22α, p21, H3K9Ac, and H3K27Ac in MOVAS treated with DMSO + Vehicle, DMSO + NaAc (120 mM), LXR-623 + Vehicle, LXR-623 + NaAc (120 mM) (48h). Band quantification is normalized to the first lane.\n< revised Figure S14H-J, new>\n➢ Fig. S14H. Lipid level in MOVAS treated with siCtrl, si-Srebf1, and si-Srebf1+NaAc by BODIPY staining (left panel) and the relative quantifications of BODIPY mean fluorescence intensity (MFI) (relative to siCtrl, right panel). (n=6; ****P<0.0001)\n➢ Fig. S14I. Immunoblot analyses of α-SMA, H3K9Ac, H3K18Ac, and H3K27Ac in MOVAS treated with siCtrl, si-Srebf1, and si-Srebf1+NaAc. Band quantification is normalized to the first lane.\n➢ Fig. S14J. Relative mRNA expression levels of Acta2 and Cdkn1a in MOVAS treated with siCtrl, si-Srebf1, and si-Srebf1+NaAc. (n=6; ****P<0.0001)\n3) Hyper-acetylation of chromatin affects expression of aging genes by opening chromatin accessibility:\nATAC-seq revealed that SREBP1 activation opens chromatin accessibility at promoters of senescence genes (including Cdkn1a, Trp53), facilitating transcription factor binding (revised Figure 6A-F).\n4) KAT2A mediates SREBP1-induced histone hyperacetylation:\n• KAT2A was upregulated at transcriptional levels in HU, SM or LXR-623-treated models, with its promoter exhibiting enhanced chromatin accessibility upon SREBP1 activation (Figure R1A -G).\n• Nuclear KAT2A accumulation was observed post-SM treatment (Figure R1H).\n• KAT2A knockdown attenuated SM-induced senescence markers and SASP genes (Figure R1I-J).\nConsistent with previous studies demonstrating SREBP1-mediated recruitment of KAT2A to activate SREBP1 and its target genes, 9 our results further suggest that KAT2A may facilitate the transcriptional regulatory functions of SREBP1.\n< Figure R1, new>\nFigure R1. GCN5 contributes to the histone hyperacetylation induced by SREBP1 activation\n(A) Heatmaps analysis of lysine acetyltransferase genes in CA of rats treated with CON or HU, MOVAS treated with CTRL or SM, MOVAS treated with DMSO or LXR-623. Kat2a (Gcn5) is highlighted.\n(B) Heatmap depicting the average signal intensity of lysine acetyltransferase genes across different groups in ATAC-seq analysis.\n(C) Relative mRNA expression levels of Kat2a in CA of rats treated with CON or HU. (n=4; **P<0.01)\n(D) Relative mRNA expression levels of Kat2a in MOVAS treated with CTRL or SM. (n=6; ****P<0.0001)\n(E) Relative mRNA expression levels of Kat2a in MOVAS treated with DMSO or LXR-623. (n=6; ***P<0.001)\n(F) Immunoblot analyses of GCN5 and α-SMA in MOVAS treated with CTRL or SM. Band quantification is normalized to the first lane.\n(G) Immunoblot analyses of GCN5 and α-SMA in MOVAS treated with DMSO or LXR-623. Band quantification is normalized to the first lane.\n(H) Representative confocal microscopy images of GCN5 (green) of MOVAS treated with CTRL or SM. Scale bar = 20 μm.\n(I) Relative mRNA expression levels of Kat2a of MOVAS transfected with different targeting siRNAs. (n=6; ****P<0.0001) si-Kat2a#1: UGUCAGAGGACGAGAUUAA; si-Kat2a#2: CAAGAAUGCCCAAGGAAUA; si-Kat2a#3: GCUGGAAGCCACUGGGAAA.\n(J) Relative mRNA expression levels of Cdkn1a, Trp53, Il6, and Ccl2 of MOVAS treated with CTRL+siCtrl, SM+siCtrl, and SM+si-Kat2a#1. (n=6; *P<0.05, ***P<0.001)\n5) SREBP1 may indirectly regulate TP53:\nOur bioinformatic analysis revealed putative SREBP1 binding sites in the TP53 promoter (Figure R2A-C), suggesting SREBP1 could transcriptionally regulate TP53 as a regulator of aging genes. However, more direct evidences for SREBP1 transcriptionally regulating aging genes still need further investigation.\nThese results substantially reinforce the SREBP1-lipogensis-acetyl-CoA-SRF/myocardin axis in our model. The Results section has been updated (page 11, lines 1-16).\n< Figure R2>\nFigure R2. SREBP1 is predicted to be involved in the regulation of TP53 expression in human, mouse, and rat\nThe predicted binding sites of SREBP1 in the promoter region (0 bp~-2000 bp) of human TP53 (A), mouse Trp53 (B), and rat Tp53 (C). Red arrows indicate the predicted binding sites. Specific information about the predicted targets is listed on the right. JASPAR 2024: https://jaspar.elixir.no/; AnimalTFDB: https://guolab.wchscu.cn/AnimalTFDB4/.\n5) The choosing of SREBP1 may be artificial according to the bulk RNA-seq data. The most significant pathway enriched is steroid biosynthesis, why the role of steroid biosynthesis and SREBP2 are neglected, note that SREBP2 is also activated in HU or SM?\nResponse:\nWe appreciate the reviewer's thoughtful comments regarding the selection of SREBP1 and the potential role of steroid biosynthesis/SREBP2. Our focus on SREBP1 was based on multiple lines of evidence:\n1) First, transcription factors (TF) enrichment analysis. The choosing of SREBP1 was not artificial. TF enrichment analysis identified SREBP1 as the most critical lipid-regulating transcription factor among 21 co-activated TFs in our RNA-seq data.\n2) Second, pathway analysis clarification. We apologize for misleading on the data presentation of KEGG analysis, of which the enrichment results were not ranked by significance. Therefore, steroid biosynthesis was not the most significant pathway according to the bulk RNA-seq. To accurately presenting our data, the KEGG-based GSEA enrichment analyses were updated (revised Figure 2A). In details, multi-pathways involved in lipid metabolism were enriched in the 47 co-activated pathways (NES>1.0) both in HU and SM models, including fatty acid metabolism (P<0.05 both in HU and SM), fatty acid biosynthesis (mainly regulated by SREBP1) (P<0.05 both in HU and SM), and steroid biosynthesis (mainly regulated by SREBP2) (P<0.05 only in SM but P=0.14 in HU) (revised Figure S4A, 2A). Further indicated that steroid biosynthesis was not the most significant pathway according to the bulk RNA-seq.\n3) Third, functional superiority of SREBP1. Firstly, in the transcriptomic data from patients with atherosclerosis, SREBP1 and its target genes showed a higher correlation with VSMC contraction (R=-0.509 vs. -0.379) and senescence-related genes (R=0.698 vs. 0.337) than SREBP2 and its target genes (revised Figure 4A, S8A-C). Secondly, the effects of SREBP1 or SREBP2 knockdown on VSMC contractile function are completely opposite, with SREBP1 knockdown significantly enhancing VSMC contractility and downregulating the expression of senescence genes, while SREBP2 knockdown impaired VSMC contraction (revised Figure 4C-D, S10A-F, S17B). Thirdly, targeted activation of SREBP1 alone (without activating SREBP2) directly induced senescence-like transformation in VSMC.\nWhile we acknowledge SREBP2's activation in HU/SM, our data consistently point to SREBP1 as the dominant regulator of VSMC senescence-like transformation. The potential role of SREBP2 activation in VSMC under simulated microgravity requires further in-depth investigation in the future.\n<revised Figure 2A, new>\n(A) KEGG based GSEA enrichment analyses for co-activated pathways of CON vs. HU and CTRL vs. SM. Normalized enrichment scores (NES) and normalized P-value (*P<0.05, ***P<0.001) are shown.\n2. Experiments design.\n1) The phenotype of SREBP1 should be also investigated in human VSMCs.\nResponse:\nBased on the reviewer’s suggestion, we have investigated the phenotype of SREBP1 in human aortic smooth muscle cells (HASMC), well-established model of human VSMC, with the following findings:\n1) SM-treated HASMC exhibited senescence-like transformation, characterized by suppression of the contractile phenotype (revised Figure S2D-E), upregulation of senescence markers (revised Figure S2E-F), and SREBP1 medicated lipid accumulation (revised Figure S6F, S7E-F), which were similar with MOVAS and RCA-SMCs.\n2) Consistent with MOVAS and RCA-SMCs, LXR-623 induced lipogenesis and senescence-like phenotypes in HASMCs by selectively activating SREBP1 rather than SREBP2 (revised Figure S11F-H, K-L, O).\nThe validation in human VSMCs fully consistent with our previous findings, strengthening the translational relevance of VSMC-SREBP1. The Results section has been updated (page 5, lines 28-30; page 7, lines 4-5; page 8, lines 2-4; page 9, lines 7-9, 22-23).\n<revised Figure S2D-F, S5F, S7D-E, S11F-H, S11K-L, S11O, new>\n➢ Fig. S2D. Relative mRNA expression levels of ACTA2, TAGLN, and CNN1 of HASMC after CTRL or SM treatment. (n=6; ****P<0.0001)\n➢ Fig. S2E. Immunoblot analyses of α-SMA, CNN1, SM22α, and P21 of HASMC after CTRL or SM treatment. Band quantification is normalized to the first lane.\n➢ Fig. S2F. Representative FACS images of HASMC after CTRL or SM treatment by SPiDER-βGal staining. Relative quantifications are shown in right. (n=6; ****P<0.0001)\n➢ Fig. S5F. Lipid level in HASMC after CTRL or SM treatment by BODIPY staining (left panel) and the relative quantifications of BODIPY mean fluorescence intensity (MFI) (right panel). (n=6; ****P<0.0001)\n➢ Fig. S7D. Immunoblot analyses of precursor-SREBP1/2 (p-SREBP1/2), and nucleic-SREBP1/2 (n-SREBP1/2) of HASMC treated with CTRL or SM. Band quantification is normalized to the first lane.\n➢ Fig. S7E. Relative mRNA expression levels of SREBF1, SREBF2, and FASN of HASMC after CTRL or SM treatment. (n=6; ***P<0.001, ****P<0.0001)\n➢ Fig. S11F. Relative mRNA expression levels of SREBF1, FASN, ACACA, and SREBF2 of HASMC treated with DMSO or LXR-623. (n=6; ***P<0.001, ****P<0.0001)\n➢ Fig. S11G. Immunoblot analyses of SREBP1 and SREBP2 of HASMC treated with DMSO or LXR-623. Band quantification is normalized to the first lane.\n➢ Fig. S11H. Lipid level in HASMC after DMSO, LXR-623 (10 μM), LXR-623 (20 μM), and LXR-623 (40 μM) treatment by BODIPY staining (left panel) and the relative quantifications of BODIPY mean fluorescence intensity (MFI) (right panel). (n=6; ****P<0.0001)\n➢ Fig. S11K. Relative mRNA expression levels of contractile genes of HASMC treated with DMSO or LXR-623. (n=6; ***P<0.001, ****P<0.0001)\n➢ Fig. S11L. Immunoblot analyses of α-SMA and SM22α of HASMC treated with DMSO or LXR-623. Band quantification is normalized to the first lane.\n➢ Fig. S11O. Relative mRNA expression levels of CDKN1A, TP53, IL6, and CCL2 of HASMC treated with DMSO or LXR-623. (n=6; ***P<0.001, ****P<0.0001)\n2) Missing controls- Fig3 (IgG), Fig4L (0 h), Fig4P (positive control such as H2O2) etc.\nResponse:\nFollowing the reviewer's suggestion, we have updated the results by adding related controls (revised Figure 1M, S1H-I, S7D, 4L, and 4P).\n3) Repeat the in vitro assays to make the N is at least 6.\nResponse:\nWe have repeated the in vitro assays to meet the requirement of N=6.\n3. Graphs. Most of the figures are of low resolution and poor quality, which should be replaced in the next version.\nResponse:\nApologize. We have replaced the figures with high-resolution.\nMinor concerns\n1. In Figure1 F, \"P21\" is marked as \"P2\".\n2. In Figure7 B, \"B-tubulin\" needs to be in the same format as above.\nResponse:\nWe greatly appreciate the reviewer’s careful reading and have corrected those mistakes.\n3. The analysis of sequence data (Fig2, 4, 6) should be moved to supplemental materials.\nResponse:\nThank you very much for your attention to our study and for your valuable suggestions. We understand your perspective. In accordance with the reviewer’s suggestion, we have moved previous Figure 2F to revised Figure S5D.\nHowever, after careful consideration, we believe that these figures (revised Figure 2B, 4G, and 6B) are crucial for presenting our key findings and conclusions. Retaining them in the main text will better facilitate the reader's understanding of our study. Here are the considerations:\n1) Presentation of Key Conclusions and Reader Convenience: Figure 2B, 4G, and 6B showcase the core discoveries of our research, which are fundamental to understanding our conclusions. Keeping these figures in the main text allows for a more direct presentation of our research ideas and key results, helping readers quickly grasp the essence of our study.\n2) Logical Coherence: These figures are closely interconnected and form a coherent narrative of this research. Moving them to the supplementary materials might disrupt this coherence, requiring readers to frequently refer back and forth between the main text and supplementary materials, which could negatively impact their reading experience.\nGiven these considerations, we propose to keep these figures in the main text. We believe this arrangement will help readers better understand and appreciate our research findings.\nReviewer #2 (Remarks to the Author):\nThe manuscript describes a novel potential role of SREBP1 in dedifferentiation and senescence of vascular smooth muscle cells through a mechanisms of epigenetic remodeling.\nThe rationale of the original study was to assess the mechanisms of vascular remodelling following changes in microgravity.\n- Reports have been published about the potential role and mechanistic pathways involving SREBP1 in atherogenesis and what effects gene expression has on EC and SMC function. However, the effect of SREBP1 in microgravity induced SMC senescence has not been described before.\nResponse:\nWe appreciate the reviewer's insightful comment. While SREBP1 has been studied in atherogenesis, our work provides the first evidence that SREBP1 regulates VSMC senescence under microgravity conditions, a critical but overlooked aspect of spaceflight-associated cardiovascular risks.\nThis novel finding bridges the gap between SREBP1’s known roles in metabolic regulation and its potential impact on cellular senescence, offering a new paradigm for spaceflight-associated vascular aging.\n- My major comment concerns the models for studying microgravity. This is not the major expertise of this reviewer but as an outsider I am surprised that this rat model is accepted as a standard to study effects of microgravity on (for example) muscle dystrophy. The same accounts for the 2-dimensional cell culture models. The animal is still under the influence of gravity (just like the cells). The animal is captured with his hindlimb in a cage which will likely also affect general metabolism, feeding patterns and subsequently maybe insulin levels (that control SREBP1). But questioning the models also questions the relevance of the outcomes for vascular pathology.\nResponse:\nWe fully understand the reviewer’s concern regarding microgravity models. While no ground-based model perfectly replicates spaceflight, the hindlimb unloading mice or rats (HU model) and two-dimensional rotation-simulated microgravity cells (SM model) are established approaches to study microgravity-associated pathophysiology, as evidenced by Jessica M Scott et al (Circ Res, 2022), Keith Siew et al (Nat Commun, 2024), Jiaxin Zhang et al (Cardiovasc Res, 2024), Zhiguo Lu et al (Bioact Mater, 2020), Li Xin Wei et al (Cell Res, 2003).10-14 Below we provide more evidences to support the reliability of microgravity models we used.\n1) First, the HU rodent model is widely accepted by National Aeronautics and Space Administration (NASA) and other space life scientists as a ground-based analogue for investigating the physiological effects induced by microgravity, particularly in cardiovascular and musculoskeletal systems. 14,15 Since the hindlimbs are fully unweighted, HU model could effectively mimic fluid shift and mechanical unloading observed in spaceflight.10 Importantly, the validated vascular effects of HU model exhibit reliable phenotypes of arterial remodeling akin to astronauts’ cardiovascular deconditioning. 16,17 Consistent with prior studies linking microgravity to spaceflight, the two-dimensional (2D) rotation-simulated microgravity cells shows confirmed microgravity-induced physiological changes, 18,19 providing a simplified system to investigate SREBP1's role in VSMC senescence under simulated microgravity.\n2) Second, we have conducted several measurements to address the potential confounders of induced by HU model, including general metabolism, feeding patterns and insulin levels. The monitored body weight, food intake, serum insulin levels showed no significant differences between HU and CON groups (revised Figure S1B and Figure R3A-B). Moreover, the 2D rotation cell models were designed to minimize stress-related confounders from circulation. 12,18,19 All these results suggest that SREBP1 changes are primarily microgravity-driven.\n3) Third, our findings are supported by consistent and remarkable phenotypic transformation of VSMC across HU rats, 2D rotation cell models of MOVAS, RCA-SMCs, and human VSMCs under simulated microgravity. VSMC dedifferentiation and senescence phenotype were validated both in vivo and in vitro (revised Figure 1-3, S1-S7), supporting the reliability of translational relevance between SREBP1 and senescence-like transformation of VSMC.\n< Figure R3A-B, new>\nFigure R3. HU treatment has no significant influence for the food intake and serum insulin in rats\n(A) Food intake (g/day) for the CON or HU treated rats. (mean±SD, n=6)\n(B) Fasting serum insulin levels for the CON or HU treated rats. (n=6)\n- The effects of gravity on organ systems is a scientific exercise of interest but the inferences for vascular disease may be limited. Also experiments have been executed to search for the translation to effects on cell metabolism in SMCs that are not affected by gravity. (For example in Figure 7). The line of thought is therefore a bit difficult to follow. The manuscript starts with differential expression studies using gravity models and it seems that in the final experiments (the regular SMC culture, non-MOVAT) confirmation is found in non MOVAT cultures while these were in fact controls in the differential expression studies.\nResponse:\nThank you for your consideration. We appreciate the reviewer’s attention to the experimental flow. Our study began with differential expression analysis in microgravity models (including HU rats and simulated microgravity models for MOVAS cells) to identify potential regulators (SREBP1) for the senescence-like transformation in VSMC. The subsequent validation in regular VSMC cultures (without microgravity stimuli) aimed to determine whether the identified mechanisms (SREBP1-medicated lipogenesis) are essential for VSMC biology and senescence. This approach helps distinguish this key mechanism (SREBP1-driven senescence) from other microgravity-specific effects.\nFurthermore, the regular SMC cultures in Figure 7 were not merely controls for differential expression but served to test the causality of SREBP1 in VSMC senescence (via knockdown). This step was essential to confirm that the observed effects are not artifacts of the microgravity model but reflect a conserved VSMC response. In addition, we further assessed the potential therapeutic effects of SREBP1 knockdown in the simulated microgravity model to validate SREBP1 as a key mechanism involved in microgravity-induced senescence-like transformation in VSMCs.\nAdditionally, while microgravity is an extreme condition, our findings have broader implications for vascular disease. Based on public RNA-seq and scRNA-seq data, our results indicated that SREBP1-mediated lipogenesis in VSMC may contribute to pathologies (VSMC phenotypic transformation and aging) in atherosclerosis.\nWhile microgravity is an extreme condition, our revised results have broader implications for vascular disease. Based on public RNA-seq and scRNA-seq data, our results indicated that SREBP1-mediated lipogenesis in VSMC may contribute to pathologies (VSMC phenotypic transformation and aging) in atherosclerosis (revised Figure 4 and S8).\nIn the revised manuscript, we have supplemented a substantial amount of additional data and reorganized the presentation of the data to enhance the logical flow.\n- If the authors suggest that SREBP1 does have an important function in cell plasticity not just under the influence of microgravity but also in general vascular pathology, then an attempt should be made to study the role of SREBP1 in SMC senescence either in a pathological animal model (such as atherosclerosis) or search for confirmation of pathways in human pathological samples (such as single cell seq of atherosclerotic specimen, data are available online).\nResponse:\nWe fully agree with the reviewer's suggestion. We have reanalyzed the previous data and supplemented it with single-cell transcriptomic sequencing data from patients with atherosclerosis to broader implications for vascular disease. The details are as follows:\nTo investigate the potential role of SREBP1 in general vascular pathology, we analyzed the expression correlation between the SREBP1 target genes and contractile (or aging) genes in the bulk transcriptome dataset of human CAs from atherosclerotic and healthy individuals (GSE100927). As shown in revised Figure 4A and S8A, SREBP1 target genes were positively correlated with aging genes and negatively correlated with VSMCs’ contractile genes.\nHowever, this result only reflects the correlations among gene sets at the bulk level of CA tissues, lacking cell-type specificity. To elucidate whether such correlations exist in VSMCs at the single-cell level, we further analyzed the mRNA expression heatmap of VSMC contractile, aging, and SREBP1-related genes across different subgroups of VSMC in the CAs from atherosclerosis patients (GSE253903) (revised Figure S8D-G). Intriguingly, VSMCs subgroups with higher expression of contractile genes exhibited lower expression of SREBP1-related and aging genes. In contrast, VSMCs subgroups with lower expression of contractile marker genes showed higher expression of SREBP1-related and aging genes (revised Figure 4B).\nConsequently, these findings indicate that the activation of SREBP1 is not only crucial for the senescence-like transformation in VSMCs induced by simulated microgravity, but also potentially impacts the phenotypic plasticity of VSMCs in general vascular pathology. The relevant results are added in revised Figure 4A-B, S8A-G and manuscript (page 8, line 22-28).\n<revised Figure S8D-G, new>\nD) Uniform Manifold Approximation and Projection (UMAP) visualization of 35 cell-clusters isolated from CA tissues of patients with atherosclerosis (GSE253903).\nE) Feature plot illustrating the expression patterns of selected cell-type-specific genes of VSMC.\nF) Feature plot illustrating the expression patterns of selected cell-type-specific genes of fibroblast.\nG) The correspondence between cell clusters and cell subgroups.\n<revised Figure 4B, new>\n(B) Heatmap depicting the average expression level of VSMC contractile genes, SREBP1 related genes, aging genes, and fibroblast genes of each VSMC or fibroblast subgroups in carotid artery from atherosclerosis patients (single-cell transcriptomic, GSE253903).\nReviewer #3 (Remarks to the Author):\nSimulated microgravity (SM) induces a senescence-like modification of carotid artery (CA) in both human and animal observations, but the underlying mechanism is not completely clear. In this manuscript, Li and colleagues reported that SM activated SREBP1-mediated lipogenesis and upregulated acetyl-CoA pool to increase histone acetylation and alter the chromatin accessibility. As a consequence, the transcription of contractile genes was reduced and the transcription of aging genes was induced, which results in a senescence-like remodeling of carotid artery. Overall, this study is interesting. However, some conclusions are not strongly supported by the data presented and some data quality needs to be improved.\nResponse:\nwe thank the reviewer for his/her positive remarks and critical criticisms, which are highly useful to improve our manuscript and to steer future studies.\nMajor concerns:\n1. In adipocytes, SREBP1c protects against adipocytes by regulating PARP1 activity (Lee et al, 2022). Muscle PARP1 inhibition can extend the lifespan of Drosophila (Guo et al, 2023). Does SREBPs-PARP1 play a role in SM and is it possible that SM promotes VSMCs senescence by activating this pathway?\nResponse:\nWe thank the reviewer for this thoughtful suggestion. While the SREBP1-PARP1 pathway plays important roles in adipocyte function (Lee et al, 2022) 20 and lifespan regulation (Guo et al, 2023) 21, our experimental data do not support its involvement in VSMC senescence. Specifically, we found no evidence of SREBP1-PARP1 interaction or PARP1 upregulation in SM induced VSMC senescence (Figure R4). This apparent discrepancy likely reflects tissue-specific differences in SREBP1 signaling and/or context-dependent activation mechanisms. Our findings suggest that SREBP1 promotes VSMC senescence through PARP1-independent pathways in this system.\n< Figure R4, new>\nFigure R4. The SREBP1-PARP1 axis is not involved in SM-induced senescence-like transformation in VSMC.\nImmunoblot analyses of SREBP1 and PARP1 after IP with anti-IgG or Pan-Ac in MOVAS treated with CTRL or SM.\n2. Activation of SREBP1 can induce increased pan-ac (including histone acetylation). But why some genes were up-regulated (aging genes) and others were down-regulated (contractile gens)? What causes selective gene regulation and different effects on chromatin accessibility?\nResponse:\nWe appreciate the reviewer’s insightful question regarding the selective gene regulation by SREBP1-induced histone acetylation. Our data and existing literature support the following explanations:\n1) Gene-specific effects of histone acetylation:\nWhile histone acetylation generally promotes chromatin accessibility, its functional outcomes are highly context-dependent. 22-24\n2) ATAC-seq evidence:\nIn our study, histone acetylation increased chromatin accessibility at aging/SREBP1-targeted genes (e.g., Srebf1, Fasn, Acly, Cdkn1a, and Trp53) but decreased it at contractile genes (e.g., Acta2, Tagln, and Cnn1), suggesting locus-specific regulation (revised Figure 6E-F).\n3) Proposed mechanism:\n⚫ Local chromatin environment (e.g., pre-existing epigenetic marks or DNA motifs) determines regional acetylation response.\n⚫ Locus-specific recruitment of transcriptional regulators (activators vs. repressors)\n⚫ Dynamic competition between epigenetic modifying complexes (acetyltransferases and deacetylases)\nWe acknowledge that further mechanistic work (e.g., ChIP-seq for key regulators) will clarify these interactions, and we will address this in future studies.\nMoreover, as LXR-623 induced acetylation of addition 34 proteins, why authors only focused on histone acetylation? Based on the model presented in Figure 8, the authors assumed that the induction of histone acetylation is the main cause for elevated expression of aging genes. But there is no data showing it is acetylation of histones but not other proteins. For Figure 5: This study should be performed under SM condition.\nResponse:\nWe appreciate this important methodological question. Our focus on histone acetylation was based on three key considerations:\n1） Biological precedence:\nHistone acetylation is a well-established epigenetic mechanism directly regulating gene expression 24-27, with multiple studies demonstrating its pro-senescence effects.28-30\n2） Direct experimental evidence:\nDaIC (specific histone acetylation inducer) recapitulated the senescent phenotype (revised Figure 5L-M, S14C-G).\n3） Technical rationale:\nWhile we observed many acetylated proteins, their functional relevance in senescence remains largely uncharacterized.\nWe prioritized histone acetylation due to its canonical role in transcriptional control, and our current data strongly support histone acetylation as the primary driver. We agree that non-histone acetylation may contribute, and this will be investigated in future studies.\nAdditionally, for Figure 5, we have supplemented the IP-MS data for VSMC in the CTRL and SM groups in revised Figure S13 and Table S4. SM treatment also induced protein hyperacetylation in MOVAS (revised Figure S13D-F). And these hyperacetylated proteins were significantly enriched in the regulation of gene expression terms, such as nucleosome, chromatin DNA binding, and nucleosome assembly (revised Figure S13G, Table S4). These pathways are intimately linked to histone H3 hyperacetylation, which was further confirmed by the increased level of H3K9Ac, H3K18Ac, and H3K27Ac in SM treated group, compared with CTRL (revised Figure S13H-J). The Results section has been updated (page 10, lines 16-24).\n< revised Figure S13D-J, new>\nD) Representative immunofluorescence images of Pan-Ac (green) of MOVAS treated with CTRL or SM. Scale bar = 100 μm.\nE) The Venn diagram of acetylated proteins (identified by IP-MS) in MOVAS treated with CTRL or SM.\nF) Heatmap of acetylated proteins (identified by IP-MS) in MOVAS treated with CTRL or SM.\nG) Gene ontology analysis of the acetylated proteins which have higher signals in SM group compared with CTRL group.\nH) Immunoblot analyses of H3K9Ac, H3K18Ac, and H3K27Ac in MOVAS treated with CTRL or SM. Band quantification is normalized to the first lane.\nI) Representative immunofluorescence images of H3K9Ac (red) of MOVAS treated with CTRL or SM. Scale bar = 100 μm.\nJ) Representative immunofluorescence images of H3K27Ac (green) of MOVAS treated with CTRL or SM. Scale bar = 100 μm.\n3. Several conclusions in this manuscript are not well supported by the data. Many data show only correlation but no causal relationship can be derived. For example, Figure 6J showed reduced nuclear translocation of SRF and MYOCD by LXR-623. But this result cannot support the conclusion that “The reshaped chromatin accessibility further decreased the nuclear translocation of SRF and MYOCD (a transcriptional coordinator of SRF), which partially contributed to transcriptional inactivity of SRF (Figure 6J).”\nResponse:\nWe thank the reviewer for highlighting this important point. We acknowledge that Figure 6J alone cannot definitively prove that chromatin accessibility changes directly impair SRF/MYOCD nuclear translocation. In the revised manuscript, we have tempered our conclusions to reflect the correlative nature of these observations.\nMoreover, to strengthen our model, we now cite additional data:\n1) ATAC-seq:\nContractile gene promoters (enriched for SRF-binding CArG boxes) showed reduced accessibility upon SM treatment (revised Figure 6E), paralleling SRF/MYOCD nuclear depletion (revised Figure 6J, S16F-G).\n2) Literature support:\nSRF activity is known to depend on chromatin openness, making our proposed mechanism plausible.31\nWhile we cannot rule out alternative mechanisms, our current data consistently support a role for SREBP1-driven chromatin remodeling in suppressing SRF/MYOCD function. Future studies with SRF rescue experiments or ChIP-seq will further validate this hypothesis.\n< revised Figure S16F-G, new>\n(A) Representative immunofluorescence images of SRF (red) of MOVAS treated with CTRL or SM. Scale bar = 20 μm.\n(B) Representative immunofluorescence images of MYOCD (red) of MOVAS treated with CTRL or SM. Scale bar = 20 μm.\nFigure 5: There is no evidence showing “Upregulated Ac-CoA pool is pivotal in the SREBP1-activation triggered senescence-like transformation of VSMCs.” The data presented are correlation between increased Ac-CoA, protein acetylation, and senescence phenotype. No causal relationship can be obtained from current data. Since ACSS2 and ACLY-mediated acetyl CoA plays a pivotal role in VSMCs senescence induced by SREBP1 activation, what is the effect of knockdown of ACSS2 and ACLY in SM-treated VSMCs? Can addition of Ac-CoA (NaAc) rescue the phenotype caused by SREBP1 knockdown?\nResponse:\nWe sincerely appreciate the reviewer's insightful comments regarding the need to establish causality between acetyl-CoA metabolism and VSMC senescence. In response to these concerns, we have conducted additional experiments that provide compelling evidence for this mechanistic relationship:\n1) ACLY/ACSS2 (mediating the synthesis of acetyl-CoA) Knockdown Studies in SM-treated VSMC (new data, revised Figure S15J-L):\n⚫ Reduced SREBP1 and P21 expression.\n⚫ Attenuated lipid accumulation and β-Gal activity.\n2) Sodium acetate (NaAc) supplementation in SREBP1-knockdown VSMC (new data, revised Figure S14H-J):\n⚫ Induced intracellular lipid accumulation.\n⚫ Increased H3 acetylation levels (H3K9Ac, H3K18Ac, and H3K27Ac).\n⚫ Offset the disrupted expression of Acta2 and Cdkn1a by SREBP1-knockdown.\n3) Mechanistic Validation:\n⚫ Pharmacological elevation of acetyl-CoA levels (NaAc) and histone hyperacetylation (deacetylase inhibitor, DaIC) both recapitulated the senescence phenotype (revised Figure 5, S13-S15)\n⚫ These findings confirm the sufficiency of the acetyl-CoA-histone acetylation axis in driving cell senescence and VSMC pathology, which also confirmed by the recently reports from Shota Yamauchi et al (Sci Adv, 2024 Oct), Yann Grobs et al (Sci Transl Med, 2024 Dec), Hao Wu et al (Circ Res, 2024 Dec). 28,32,33\nWe thank the reviewer for prompting these important clarifications, which have significantly strengthened our study. These new experimental results provide direct evidence supporting our conclusion that upregulated Ac-CoA pool contributes to SREBP1-activation triggered senescence-like transformation of VSMC. We have revised the manuscript text to more accurately reflect these relationships (pages 10-11).\n<revised Figure S15F-L, new>\n(M) Relative mRNA expression levels of Acly of MOVAS transfected with different targeting siRNAs. (n=6; ****P<0.0001)\n(N) Immunoblot analysis of ACLY of MOVAS transfected with si-Acly #2. Band quantification is normalized to the first lane.\n(O) Relative mRNA expression levels of Acss2 of MOVAS transfected with different targeting siRNAs. (n=6; ****P<0.0001)\n(P) Immunoblot analysis of ACSS2 of MOVAS transfected with si-Acss2 #2. Band quantification is normalized to the first lane.\n(Q) Lipid level in MOVAS treated with CTRL+siCtrl, SM+siCtrl, SM+si-Acly, and SM+si-Acss2 by BODIPY staining (left panel) and the relative quantifications of BODIPY mean fluorescence intensity (MFI) (right panel). (n=6; **P<0.01, ***P<0.001, ****P<0.0001)\n(R) Senescence level in MOVAS treated with CTRL+siCtrl, SM+siCtrl, SM+si-Acly, and SM+si-Acss2 by SPiDER-βGal staining (left panel) and the relative quantifications of SPiDER-βGal mean fluorescence intensity (MFI) (right panel). (n=6; **P<0.01, ***P<0.001, ****P<0.0001)\n(S) Immunoblot analyses of SREBP1 and P21 in MOVAS treated with CTRL+siCtrl, SM+siCtrl, CTRL+ si-Acly, SM+si-Acly, CTRL+ si-Acss2, and SM+si-Acss2. Band quantification is normalized to the first lane.\n< revised Figure S14H-J, new>\n➢ Fig. S14H. Lipid level in MOVAS treated with siCtrl, si-Srebf1, and si-Srebf1+NaAc by BODIPY staining (left panel) and the relative quantifications of BODIPY mean fluorescence intensity (MFI) (relative to siCtrl, right panel). (n=6; ****P<0.0001)\n➢ Fig. S14I. Immunoblot analyses of α-SMA, H3K9Ac, H3K18Ac, and H3K27Ac in MOVAS treated with siCtrl, si-Srebf1, and si-Srebf1+NaAc. Band quantification is normalized to the first lane.\n➢ Fig. S14J. Relative mRNA expression levels of Acta2 and Cdkn1a in MOVAS treated with siCtrl, si-Srebf1, and si-Srebf1+NaAc. (n=6; ****P<0.0001)\n4) Figure 7H-O: It is good to see authors examined the role of SREBP1 in SM-induced senescence phenotype in vivo. However, it is recommended to examine additional factors, including P21 staining, β gal staining, the transcription of contractile genes and aging genes.\nResponse:\nWe appreciate the reviewer's suggestion to strengthen our in vivo characterization. In response, we have performed additional analyses with the transcriptional expression of contractile and aging genes, as well as immunofluorescence staining for P21 and Pan-Ac in the carotid arteries of the CON and HU groups following Lv-sh-Ctrl or Lv-sh-Srebf1 treatment. In line with attenuated structural remodeling, Lv-sh-Srebf1 treatment partially restored the disordered transcription of Srebf1, Acta2, Tagln, Cdkn1a, and Tp53, as well as the expression of P21 and Pan-Ac in the carotid artery treated with HU (revised Figure 7M-N).\nThese new data provide a more complete picture of how SREBP1 inhibition attenuates both molecular and phenotypic aspects of vascular senescence. The results have been incorporated into the revised manuscript (page 13, line 23-26).\n< revised Figure 7M-N, new>\n(M) Relative mRNA expression levels of Srebf1, Acta2, Tagln, Cdkn1a, and Tp53 in carotid artery of rats treated with Lv-shCtrl+CON, Lv-shCtrl+HU, Lv-sh-Srebf1+CON, and Lv-sh-Srebf1+HU. (n=4; *P<0.05, **P<0.01, ***P<0.001)\n(N) Representative immunofluorescence images of Pan-Ac (red), and P21 (green) in carotid artery of rats treated with Lv-shCtrl+CON, Lv-shCtrl+HU, Lv-sh-Srebf1+CON, and Lv-sh-Srebf1+HU. Scale bar = 200 μm or 100 μm (zoom in panel).\n5) Figure S2E-S2F: Figure S2E show that SM inhibits the proliferation of MOVAS. Figure S2F show that SM killed MOVAS cells. The authors should show the detail of experiments. Whether the SM induced cell senescence or killed young cells? How to exclude the role of apoptosis on transformation phenotype and functional decline of vascular?\nResponse:\nThank you for the reviewer's suggestion.\n1) We have incorporated the details of experiments as suggested by the reviewer in revised manuscript (page 6, line 9-11).\n2) To determine whether SM treatment induces cellular senescence or directly kills young cells, we further collected cells that were shed into the supernatant following CTRL or SM treatment. As shown in revised Figure S2M-N, cells that were shed following SM treatment exhibited stronger apoptotic signaling (revised Figure S2M) and higher expression of senescent marker genes (revised Figure S2N), which suggest that SM treatment induces cellular senescence rather that killing young cells. The related descriptions were added in revised manuscript (page 6, line 11-13).\n3) We appreciate the reviewer's important question regarding the potential role of apoptosis in VSMC phenotypic transformation. Our response addresses this concern through three key points:\n⚫ In vascular pathologies (e.g., neointima formation, atherosclerosis), VSMC apoptosis and senescence frequently coexist and jointly contribute to disease progression. 34-38 This supports the biological plausibility of both processes occurring simultaneously in our model.\n⚫ Apoptosis and cellular senescence share several common mechanisms, including P53 activation, mitochondrial dysfunction, oxidative stress, activated autophagy, and the involvement of interconnected signaling networks. 39-42\n⚫ Our study focused on VSMC senescence in microgravity-induced vascular remodeling. While senescence appears key, apoptosis may also contribute, the role of apoptosis will be examined in future work.\n< revised Figure S2M-N, new>\n(M) Representative apoptotic FACS images of the supernatant cells in CTRL or SM group by Annexin-FITC/PI staining.\n(N) Relative mRNA expression levels of Cdkn1a and Trp53 of MOVAS in CTRL, SM, or SM (supernatant) group. (n=6; ****P<0.0001)\n6) It has been established that Ac-CoA is compartmentalized inside cells, including cytoplasm, mitochondria and nucleus. Histone acetylation increases could be more associated with nucleus acetyl-CoA. I am curies what pools of acetyl-CoA were significantly enhanced in MOVAS after both LXR-623 and SM treatments?\nResponse:\nWe thank the reviewer for raising this important point regarding subcellular acetyl-CoA compartmentalization. In response, we performed cellular fractionation experiments in MOVAS cells and found that both LXR-623 and SM treatments specifically increased acetyl-CoA levels in the cytoplasmic and nuclear compartments, while mitochondrial acetyl-CoA pools remained unchanged (revised Figure S13B-C). These results suggest that the observed histone hyperacetylation is likely driven by nuclear acetyl-CoA accumulation, consistent with the known compartmentalization of acetyl-CoA metabolism. The relevant results have been added to revised Figure S13B-C and manuscript (page 10, line 9-11).\n< revised Figure S13B-C, new>\n(B) Relative levels of Ac-CoA of cytoplasm, nucleus, or mitochondria in MOVAS treated with DMSO or LXR-623. (n=6; **P<0.01, ***P<0.001)\n(C) Relative levels of Ac-CoA of cytoplasm, nucleus, or mitochondria in MOVAS treated with CTRL or SM. (n=6; **P<0.01)\nMinor concerns:\n1. The quantification of Western blots is inaccurate. For example, Figure 3F (n-SREBP1), 5J (SMA), 5L (p21), 6J (MYOCD, no band but have grayscale) value, etc. The authors should also mark the non-specific bands in figure 3C, 4F, M.\nResponse:\nThank you for this suggestion. We have double-checked and corrected the quantification data for the Western Blots. The non-specific bands in Figure 3C, 4F, 4M have been marked in revised Figure 3E, 4F, and 4M.\n2. Page 5, line 16, line 19: should be Figure 1B-C, Figure 1D-F.\nResponse:\nThank you for your reminder. We have checked it.\n3. The role of acetyl-CoA and ACSS2 in histone acetylation and senescence has been reported (PMID: 33917812; 34183849). The authors should discuss this point in their work.\nResponse:\nWe sincerely appreciate the reviewer's valuable suggestion regarding the established role of acetyl-CoA/ACSS2 in histone acetylation-mediated senescence. We have explicitly acknowledged the seminal work by referencing both suggested publications (PMID: 33917812; 34183849) and related studies (PMID: 39661707, 39513269), as shown in the revised Discussion. 32,33,43,44\nNotably, our results not only confirm this fundamental mechanism in VSMCs, but also provide novel insights by identifying microgravity as a new physiological regulator of the ACLY/ACSS2/Ac-CoA axis and stablishing the first connection between this metabolic pathway and vascular adaptation to microgravity.\nThe modified text appears in the Discussion section (page 16, line 5-13) of the revised manuscript. We believe these additions have strengthened the conceptual framework of our study while properly crediting prior discoveries.\n4. The key materials and methods used in this study should be included in the main text instead of putting in the supplementary materials.\nResponse:\nIn response to the reviewer's comment, we have revised the main manuscript to include key materials and methods.\n5. There is no description of how authors performed analysis for GSEA based activated TF enrichment analysis.\nResponse:\nThank you for your reminder. We have incorporated the GSEA based activated TF enrichment analysis into the revised methods (page 24, line 22-24).\n6. Fig. 1F: p2 should be p21.\nResponse:\nThank you for your reminder. We have checked it.\nReviewer #4 (Remarks to the Author):\nI 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.\nResponse:\nWe sincerely appreciate the time and expertise of both reviewers in evaluating our manuscript.\nReviewer #5 (Remarks to the Author):\nThe manuscript titled “SREBP1-mediated lipogenesis promotes dedifferentiation and senescence of vascular smooth muscle cells through epigenetic remodeling” by Li et al. aims to mechanistically explain the phenomenon of VSMCs senescence and senescence-like modifications in carotid artery upon simulated microgravity. Authors use in vitro, ex vivo and in vivo models together with a range of molecular biology, biochemistry, imaging and omics techniques to demonstrate that VSMC senescence occurs through cellular epigenetic remodeling caused by SREBP1-mediated lipogenesis.\nAdvances in space exploration justify this study and the overall need to better understand the detrimental effect of microgravity on human body. It is evident that Authors have substantial experience in utilizing the technique of simulated microgravity. I appreciate the amount of work that has been done by Authors.\nEven though this complex study provides substantial amount of data, presented results do not support the entireness of tested hypothesis. The Authors provide data that demonstrates the involvement of SREBP1 in senescence-like processes in carotid arteries, however, evidence that lipogenesis promotes senescence via epigenetic remodeling is limited. Also, the manuscript must go through an extensive proofreading process first as the text for both the main and supplementary part has a lot of typos and mistakes (page 3 line 14 tunica media vasorumta, Fig1L ‘cell differention’, etc).\nResponse:\nWe sincerely appreciate the reviewer’s thoughtful evaluation and constructive suggestions.\nTo strengthen the evidence that lipogenesis promotes senescence via epigenetic remodeling, we have performed additional experimental validation and discussed the potential limitations as follows:\n1) Added new data demonstrating histone acetylation in VSMC under SREBP1 activation and simulated microgravity (revised Figure S13, S14, and S15), showing direct correlation between lipid accumulation and epigenetic changes.\n2) Clarified limitations in the Discussion as follows: the other lipid-mediated pathways or acetylated protein may contribute to VSMC senescence, and future studies will dissect their interplay with epigenetics (page 16, lines 24-26).\n3) Cited supporting literature to demonstrate lipogenesis as epigenetic modulators in cell senescence and VSMC pathology in the Discussion (page 15, lines 19-29; page 16, lines 5-13).\nWe apologize for typographical and spelling errors. The manuscript has now been thoroughly checked, the typographical and spelling errors have been corrected.\nMajor issues:\n1. Authors do not provide enough details for some of the assays essential for hypothesis testing. The major concern is around “Absolute Quantitative Lipidomics”. Authors do not provide any information or references around chromatographic columns they used, solvent gradients for lipid separation, list of internal standards and their quantities, mass spectrometry parameters, and overall, a method that was used for absolute lipid quantification making this part of the study non-reproducible. If the targeted lipid analysis was used, the authors must describe how absolute lipid quantities were estimated. How did Authors confirm (Table S2) the fatty acid composition including the exact fatty acid sn-position of PC lipids? How abundances of isomeric lipid species were estimated (e.g., TG46:1) while these molecules coelute as one peak (RT 8.61 min), however, Authors managed to quantify individual isomers? Are the numbers in Table S2 expressed as absolute concentrations? If so, what are the values (nM, ng)? I assume that these values were normalized to some amount of material taken for the analysis. If so, what is it – mg pf protein or wet sample weight? If absolute quantitative lipid analysis was performed, what was the utility of lipid ELISA assay, especially for TG and FA. How comparable is the data from the mass spectrometry vs ELISA?\nResponse:\nWe thank the reviewer for raising these important points. In response to the reviewer’s request, we have provided additional details regarding the lipidomics in the Methods section of the revised manuscript (page 24, line 27 to page 25, line 25). The details are as below:\n1) Chromatography:\n• Column: C18 column (Phenomenex, Kinetex C18, 2.1 × 100 mm, 2.6 μm).\n• Solvent gradients for lipid separation: For C18 separation, the column temperature was set at 45 °C. Mobile phase A: 70% acetonitrile + 30% H2O+5mM ammonium acetate, mobile phase B: IPA solution. A gradient (20% B at 0 min, 60% B at 5 min, 100% B at 13 min, 20% B at 13.1-17 min) was then initiated at a flow rate of 0.35 mL/min.\n• List of internal standards and their quantities：\nFor the standards, we used SPLASH™ LIPIDOMIX™ Mass Spec Standard (10 μL, AVANTI, 330707-1EA), containing 15:0-18:1(d7) PC (160 μg/mL); 15:0-18:1(d7) PE (5 μg/mL); 15:0-18:1(d7) PS (5 μg/mL); 15:0-18:1(d7) PG (30 μg/mL); 15:0-18:1(d7) PI (10 μg/mL); 15:0-18:1(d7) PA (7 μg/mL); 18:1(d7) LPC (25 μg/mL); 18:1(d7) LPE (5 μg/mL); 18:1(d7) Chol Ester (350 μg/mL); 18:1(d7) MG (2 μg/mL); 15:0-18:1(d7) DG (10 μg/mL); 15:0-18:1(d7)-15:0 TG (55 μg/mL); 18:1(d9) SM (30 μg/mL); (d7) Cholesterol (100 μg/mL).\n2) Mass spectrometry parameters:\n• Instrument: 6500+QTRAP (AB SCIEX) was performed in positive and negative switch mode.\n• Parameters: The ESI positive source conditions were as follows: Source temperature: 400 °C; Ion Source Gas1 (GS1): 50 Ion Source Gas2 (GS2): 55; Curtain Gas (CUR): 35; IonSpray Voltage (IS): +3000 V; The ESI negative source conditions were as follows: Source temperature: 400 °C; Ion Source Gas1 (GS1): 50; Ion Source Gas2 (GS2): 55; Curtain gas (CUR): 35; IonSpray Voltage (IS): -2500 V.\n3) Quantitative data processing:\n• Multiple Reaction Monitoring method was used for mass spectrometry quantitative data acquisition and Sciex OS was used for quantitative data processing by Shanghai Applied Protein Technology Co., Ltd. (Shanghai, China).\n• Normalization: Data expressed as ng/g sample weight.\nRegarding the choice of lipids detection methods, the samples analyzed by lipidomics and ELISA were distinct. Lipidomics was performed to detect lipid distribution in the CA tissues of CON and HU rats (revised Figure 2F-K, S6A-D). However, ELISA was used to measure TG and FA levels in RCA-SMCs after CTRL and SM treatment (revised Figure S5C-D). Notably, the results of these two methods for lipids detection further corroborate the activation of lipid biosynthesis indicated by the RNA-seq data in the HU and SM models (revised Figure 2A-B, S3-S4).\n2. Cell proliferation cannot be estimated using cell viability assay. Why didn’t authors use the gold standard Ki-67 as the proliferation marker?\nResponse:\nBased on the reviewer’s suggestion, we have performed Ki-67 staining assays to assess cell proliferation. As shown in the revised Figure S2K, SM treatment significantly inhibited VSMC proliferation compared with the CTRL group. We have removed the CCK8 assay data to avoid misinterpretation. The Results section has been updated (page 6, lines 9-11).\n<revised Figure S2K, new>\n(K) Representative immunofluorescence images of Ki-67 (green) of MOVAS after CTRL or SM treatment. Scale bar = 100 μm. Relative quantifications are shown in right. (n=6; ***P<0.001)\n3. For the 2D SM cell models, it is not clear what the control was. Supplementary page 4 line 20 – “Cells without rotation (1 g) were cultured in the same manner as the control group.” If one guided by corresponding Figure S2A, cultured cells in control group (CTRL) are covered with small layer of culture medium allowing cells to grow under normoxic conditions, while in case of SM cell model, the bottle is filled with media that will force cells to be under more hypoxic conditions as well as experience increased pressure due to the amount of liquid on top of them. If this is the case, observed lipid droplet accumulation and cell viability and senescence could be attributed to the experimental setup.\nResponse:\nWe thank the reviewer for raising this important methodological consideration and appreciate the opportunity to clarify this critical experimental detail.\n1） To clarify, both the SM and control groups were cultured with identical medium volumes (fully filled bottles) to ensure consistent oxygenation and eliminate hydrostatic pressure differences, in line with established microgravity simulation protocols. 18,19\n2） Transcriptomic analysis confirmed comparable oxygenation between groups, as evidenced by no significant HIF-1 pathway activation (P=0.39, FDR=0.78; Figure R5A-B).\n3） We have corrected Figure S2A to accurately reflect these matched conditions, confirming that the observed lipid accumulation and senescence phenotypes are attributable to simulated microgravity rather than hypoxia or pressure artifacts.\n< Figure R5A-B, new>\nFigure R5. The HIF-1 signaling pathway is not significantly activated in MOVAS after SM treatment.\n(A) GSEA plots showing normalized enrichment scores (NES) and normalized P-value for HIF-1 signaling pathway from RNA-Seq (CTRL vs. SM).\n(B) The overview of the HIF-1 signaling pathway from RNA-Seq (CTRL vs. SM), with red indicating significantly upregulated genes and green indicating significantly downregulated genes.\n< revised Figure S2A, modified>\n(A) Schematic diagram to generate simulated microgravity (SM) model of cells.\n4. Authors claim that SM in rat carotid artery (CA – confusing acronym as CA is used for citric acid in the manuscript) using HU model causes senescence-like remodeling in VSMCs by showing an increase in relative mRNA levels of genes associated with senescence (Fig 1). Carotid artery contains other cell types (endothelial cells, fibroblasts, dendritic cells, etc). There is no data presented showing that the signal increase is coming specifically from VSMCs alone.\nResponse:\nWe thank the reviewer for highlighting these important issues.\n1) To avoid confusion, we have replaced \"CA\" with \"carotid artery\" throughout the manuscript and reserved \"citric acid\" for metabolic contexts.\n2) We acknowledge that bulk mRNA data from carotid arteries (Figure 1) could reflect contributions from non-VSMC, although VSMC represent the mainly cell type in carotid arteries. 45 To address this, we conducted multiplex immunofluorescence staining on carotid artery of HU rats, showing that the upregulated expression of P21 and SREBP1 induced by HU were predominantly localized in VSMC of vascular media (marked by α-SMA), rather than endothelial cells (marked by CD31) (revised Figure 1H, 3D).\n3) Moreover, human umbilical vein endothelial cells (HUVECs) (a canonical model of vascular endothelial cells) were cultured for further in vitro assays. After 48 hours of SM treatment, HUVECs did not exhibit pronounced senescence-like phenotypes observed in VSMCs, including increased cell size, upregulation of aging or SASP markers, or activation of SREBPs (revised Figure S2G-I, S7G). These results indicate that endothelial cells and their SREBP1 signaling contribute minimally to vasculature aging under microgravity.\nBesides, we have clarified the limitation in the Discussion (page 16, line 18-22) noting that other non-VSMC cells (including fibroblasts, pericytes, and immune cells) may contribute to the microgravity-induced vascular remodeling, which require further investigation in future studies.\n<revised Figure S1H, new>\n(H) Schematic diagram of the carotid artery cross-section.45 Endothelial cells primarily constitute the intima of the carotid artery. Vascular smooth muscle cells (VSMC) are the main component of the media.\n<revised Figure 1H, 3D, new>\n➢ Fig. 1H. Representative immunofluorescence images of P21 (red), CD31 (cyan), and α-SMA (green) in the carotid artery from CON and HU rats. Intensity traces (offset white lines) are plotted at right. “L” indicates the side of vascular lumen. Scale bar = 200 μm or 100 μm (zoom in panel).\n➢ Fig. 3D. Representative immunofluorescence images of SREBP1 (red), CD31 (cyan), and α-SMA (green) in the carotid artery from CON and HU rats. Intensity traces (offset white lines) are plotted at right. “L” indicates the side of vascular lumen. Scale bar = 200 μm or 100 μm (zoom in panel).\n<revised Figure S2G-I, S7G, new>\n➢ Fig. S2G. Representative FACS images of HUVEC after CTRL or SM treatment. FSC=forward scatter; SSC=side scatter.\n➢ Fig. S2H. Relative mRNA expression levels of CDKN1A, CDKN2A, TP53, IL6, and CCL2 of HUVEC after CTRL or SM treatment. (n=6)\n➢ Fig. S2I. Immunoblot analyses of P21 of HUVEC after CTRL or SM treatment. Band quantification is normalized to the first lane.\n➢ Fig. S7G. Relative mRNA expression levels of SREBF1, SREBF2, FASN and ACACA of HUVEC after CTRL or SM treatment.\n5. My understanding that the heatmap (Fig 2F) is derived from the targeted lipid analysis data in Table S2. If so, why do authors use just the data from only 3 HU rats, while the dataset in Table S2 has lipid data of 4 HU rats. What was the reason to omit one of the replicates? Same for Fig S5C (PCA analysis). And what was used as QC – not clear.\nResponse:\nWe apologize for our oversight. We have now presented all the data in the revised Figure 2F-K, S6A-D. The quality control (QC) samples are a composite prepared by pooling equal volumes of all the samples to be tested. The relevant descriptions have been added to the methods (page 24, lines 31-33). The newly supplemented data do not affect the conclusions of the study.\n<revised Figure S6A-D, modified>\n(A) Summary of all the lipid molecules distributed across 21 classes detected by absolute quantitative lipidomics. Specific number is shown above the columns.\n(B) The Log10-transformed lipid concentration (mg/g tissue) of all the carotid artery samples from CON and HU rats.\n(C) PCA analysis of lipidomics of the carotid artery samples from CON and HU rats. The quality control (QC) samples are a composite prepared by pooling equal volumes of all the samples to be tested.\n(D) Heatmap analysis of lipidomics of the carotid artery samples from CON and HU rats.\n6. Quality of Western Blots. Fig 4F – loading control for n and p-SREBP2 is saturated, too much material was loaded. Same for Fig 5G, Fig S13D – I am not sure if quantification is correct. Poor WB quality – Fig Fig S11I, Fig S13C.\nResponse:\nThank you for the suggestion. We have replaced the Western Blots to enhance the quality of the article.\n7. Given the fact that SM has profoundly different effect on fatty acid synthesis (Fig 2B) between the rat HU model and MOVAS SM model (e.g. Fasn difference 1.9 fold change increase in rat model vs 0.86 in MOVAS SM, Acly – 2.5 vs 0.67), what was the rational to move away from the HU rat model and test the hypothesis on MOVAS SM cell model? It is easy to test the drug in tissue culture, however, what grounds do Authors have to claim that similar mechanisms occur in vivo?\nResponse:\nWe appreciate the reviewer's careful examination of the data. We clarify that all fold changes (FC) in Figure 2B and 4I are Log2-transformed values (now explicitly noted in the figure legend; page 33, line 31 and page 35, line 31), where positive values (>0) indicate upregulation and negative values (<0) downregulation. Thus, both the HU-rat (FASN Log2FC=1.9) and SM-MOVAS (Log2FC=0.86) models show consistent upregulation of FASN—a pattern corroborated by the heatmap’s overall concordance in lipogenic gene expression trends between models. While the magnitude differs (likely reflecting cell-autonomous vs. systemic responses), the conserved directional changes support MOVAS as a valid model for mechanistic studies, with key findings (SREBP1 activation and histone hyperacetylation) validated across both systems.\nOther comments:\n1. P 5 L16 – missing Fig number.\nResponse:\nWe have corrected the Figure number.\n2. P 5 L 15-16 – quantification of lumen area is not present. Authors claim that the lumen area is unchanged, however, there is no difference in the cross-section diameter of RCCA, although cIMT in HU is significantly increased. Please comment.\nResponse:\nWe thank the reviewer for raising this important point. Our vascular ultrasound measurements (revised Figures 1D and S1G) demonstrate that while carotid intima-media thickness (cIMT) was significantly increased in HU rats, the lumen diameter remained unchanged compared to controls. This apparent discrepancy likely reflects two concurrent processes: (1) expansion of the vessel wall due to senescence-like VSMC enlargement, and (2) compensatory lumen preservation mediated by reduced vascular contractility in the HU model, consistent with previous reports of vascular remodeling under microgravity. 10,16,46-49\n3. P5 L20 and Fig 1F – there is no corresponding image of SMA staining that can confirm that the p21 signal comes from SMCs\nResponse:\nThank you for your reminder. Further immunofluorescence assays were conducted and clarified that the upregulated expression of P21 induced by HU were predominantly localized in VSMC (marked by α-SMA), rather than endothelial cells (marked by CD31). The relevant results have been incorporated into the revised manuscript (page 5, line 18-19; page 7, line 28-29) and revised Figure 1H, 3D.\n<revised Figure 1H, 3D, new>\n➢ Fig. 1H. Representative immunofluorescence images of P21 (red), CD31 (cyan), and α-SMA (green) in the carotid artery from CON and HU rats. Intensity traces (offset white lines) are plotted at right. “L” indicates the side of vascular lumen. Scale bar = 200 μm or 100 μm (zoom in panel).\n➢ Fig. 3D. Representative immunofluorescence images of SREBP1 (red), CD31 (cyan), and α-SMA (green) in the carotid artery from CON and HU rats. Intensity traces (offset white lines) are plotted at right. “L” indicates the side of vascular lumen. Scale bar = 200 μm or 100 μm (zoom in panel).\n4. No data on mRNA expression of Ascc1 or Dgat1 was presented. ASCC1 is mitochondrial protein and can contribute to Acetyl-CoA pool, DGAT1 can play a compensatory role if DGAT2 is absent or inhibited. It is surprising to see an accumulation of lipid droplets filled with TG (based on your lipidomics data), however, Dgat2 expression is downregulated in HU or SM (Fig 2B). How would you explain higher TG biosynthesis without corresponding upregulation of Dgats? Decreased TG lipolysis? Need data showing this if so…\nResponse:\nWe appreciate the reviewer’s inquiry. In response, we have supplemented our data by examining the mRNA expression of Dgat1 and Acss1 (revised Figure R6A-B and S15D-E). The results show no significant changes in Dgat1 or Acss1 expression in HU-treated CA or SM-treated MOVAS, suggesting that compensatory upregulation of these genes does not account for the observed TG accumulation.\nTo further explore potential mechanisms, we analyzed the expression of key lipolysis-related genes in our transcriptome data (revised Figure R6C-D). Most lipolytic genes remained unchanged in HU- or SM-treated groups, indicating that impaired TG breakdown is unlikely to be the primary driver of lipid droplet accumulation.\nGiven these findings, we propose that the increased TG levels are primarily driven by enhanced de novo lipogenesis rather than suppressed lipolysis. Although Dgat2 expression was downregulated, the elevated availability of substrates (acetyl-CoA and fatty acids) may stimulate DGATs enzymatic activity, thereby promoting TG synthesis even in the absence of transcriptional upregulation of Dgats.\n<revised Figure S15D-E, new>\n(A) Relative mRNA expression levels of Acss1 in MOVAS treated with CTRL or SM. (n=6)\n(B) Relative mRNA expression levels of Acss1 in CA of rats treated with CON or HU. (n=3)\n< Figure R6, new>\nFigure R6. The expression of Dgat1 and lipolysis genes remained unchanged in the HU and SM groups.\n(A) Relative mRNA expression levels of Dgat1 in carotid artery from CON and HU rats. (n=3)\n(B) Relative mRNA expression levels of Dgat1 in MOVAS after CTRL or SM treatment. (n=6)\n(C) mRNA expression (FPKM) of lipolysis genes from RNA-seq in carotid artery from CON and HU rats. (n=3, ***P<0.001)\n(D) mRNA expression (FPKM) of lipolysis genes from RNA-seq in MOVAS after CTRL or SM treatment. (n=3, **P<0.01)\n5. Fig 1K – difficult to appreciate the difference – please provide quantification for this assay.\nResponse:\nWe fully agree with the reviewer's suggestion. The relevant statistics have been incorporated into the revised Figure 1M.\n<revised Figure 1M, new>\n(M) Representative images of cell contraction assay of MOVAS after CTRL or SM treatment. Quantifications of cell contraction diameters are shown in the right panel. (n=5; ***P<0.001)\n6. P 6 L 29-32 – claimed increase in FA isn’t supported by data on Fig S5C-E (green dot for FA indicates almost no change).\nResponse:\nWe sincerely appreciate the reviewer's careful observation regarding the fatty acid (FA) data presentation. Upon re-examination, we identified that there was indeed an inadvertent figure reference error in the original manuscript. Specifically:\n1) The claim about increased FA levels was supported by data in Figure S6C-E (showing ELISA and BODIPY results from SM-treated RCA-SMCs), rather than Figure S5C-E (which displays lipidomic data from carotid artery tissues).\n2) Our revised data demonstrate:\n• In carotid arteries (revised Figure 2H), HU treatment showed increased FA content compared to CON group.\n• In RCA-SMCs (revised Figure S6C-E), SM treatment significantly elevated FA levels along with triglycerides and intracellular lipids relative to CTRL group.\nWe have carefully corrected all figure references throughout the manuscript and performed a thorough review to ensure such inconsistencies do not occur elsewhere. The current version accurately reflects our experimental findings.\n7. P 6 L 32-33 – reference to the Figure S6 does not correspond to the actual Figure S6.\nResponse:\nWe sincerely appreciate the reviewer's careful attention to this detail. We have carefully reviewed the figure references and confirmed that there was indeed an incorrect citation in the original manuscript. We have now thoroughly verified all figure references in the revised version to ensure complete accuracy.\nReferences:"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors nicely addressed all the concerns, no further comments need to be raised.", "reviewer_2": "1- The authors have significantly revised the manuscript with the addition of relevant data. My initial comments focus mainly on the validity of models used for microgravity effects and the experimental design in which the generic value of SREBF1 in smooth muscle cell transition and senescence was examined.\nThe authors argue that the chosen models for microgravity are a standard in this field of research. I understand the argument and also that studying biological transitions after microgravity change is complex and that the ideal model does not exist (unless you are in space). That does not mean that a model cannot be questioned, because the fact remains that even if the leg becomes dystrophic and undergoes changes due to e.g. lack of muscle use, that the models are still subject to gravity. This is a shortcoming that cannot be solved experimentally, but it is always good to point out in a discussion that the observations found can be explained by other physical mechanisms than \"gravity\".\n2- My other comment was about the extrapolation of the gravity model to more generic mechanisms of vascular wall diseases. This is relevant and the authors have now focused on ageing effects on the smooth muscle cell and used public datasets to show that expression changes with increasing age. This leaves the manuscript limping on 2 thoughts: the effects of microgravity on vascular wall pathology and the role of SREBF1 and the role of SREBF1 (independent of gravity) on ageing, senescence, of smooth muscle cells.\nIt is a matter of choice, but actually the most powerful evidence for causality is the execution of a knock down (and/or an intervention) in an animal model, such as the atherosclerotic mouse. But for SREBPF1 (but especially SREBF2) the necessary experiments have already been performed (example; PMID: 30354203). The authors could mention in the discussion that the effects of the gene in relation to e.g. atherosclerosis are supportive for the observed results.", "reviewer_3": "In the revised manuscript, the authors have addressed all my concerns. This manuscript is ready for publication.", "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": "Thank you for taking the time to fully revise the manuscript. I appreciate the authors' efforts in addressing the concerns I raised in my initial review. The revisions have adequately clarified the key issues, and I find the manuscript substantially improved.\nThat said, I would like to emphasize that manuscripts of this complexity and technical depth require careful drafting and thorough internal revision prior to submission. A more rigorous pre-submission review by the authors could have avoided some of the initial issues (like typos and ambiguous acronyms) and facilitated a smoother review process.\nOverall, I am satisfied with the current version and have no further major concerns."}, "author_response": "REVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors nicely addressed all the concerns, no further comments need to be raised.\nResponse:\nWe thank the reviewer for their time and valuable comments, which have significantly improved our manuscript.\nReviewer #2 (Remarks to the Author):\n1- The authors have significantly revised the manuscript with the addition of relevant data. My initial comments focus mainly on the validity of models used for microgravity effects and the experimental design in which the generic value of SREBF1 in smooth muscle cell transition and senescence was examined.\nThe authors argue that the chosen models for microgravity are a standard in this field of research. I understand the argument and also that studying biological transitions after microgravity change is complex and that the ideal model does not exist (unless you are in space). That does not mean that a model cannot be questioned, because the fact remains that even if the leg becomes dystrophic and undergoes changes due to e.g. lack of muscle use, that the models are still subject to gravity. This is a shortcoming that cannot be solved experimentally, but it is always good to point out in a discussion that the observations found can be explained by other physical mechanisms than \"gravity\".\nResponse:\nWe sincerely thank the reviewer for this insightful comment, which helps to enhance the scholarly rigor of our manuscript. We fully agree with the reviewer that it is crucial to thoughtfully discuss the limitations of the ground-based microgravity simulation models. While these models are indispensable tools for space physiology research, they indeed simulate a state of \"mechanical unloading\" rather than true zero gravity.\nAs suggested, we have now added the limitation in the Discussion section (page 16, line 25 to page 17, line 4) to explicitly address this point. In this section, we:\n1. Acknowledge the inherent limitation of the hindlimb unloading model, specifically that it operates within Earth's gravity.\n2. Discuss that the observed vascular phenotypes could be attributed to alterations in the overall mechanical environment (e.g., hemodynamic changes, altered vascular wall stress) simulated by the model, which are concomitant with gravitational changes.\n3. Suggest that future studies aboard real spaceflight missions or using complementary ground-based models will be valuable to dissect the specific contributions of these mechanical factors.\nWe believe this addition provides a more balanced and critical perspective on our findings, significantly strengthening the manuscript. We are grateful for this constructive suggestion.\n2- My other comment was about the extrapolation of the gravity model to more generic mechanisms of vascular wall diseases. This is relevant and the authors have now focused on ageing effects on the smooth muscle cell and used public datasets to show that expression changes with increasing age. This leaves the manuscript limping on 2 thoughts: the effects of microgravity on vascular wall pathology and the role of SREBF1 and the role of SREBF1 (independent of gravity) on ageing, senescence, of smooth muscle cells.\nIt is a matter of choice, but actually the most powerful evidence for causality is the execution of a knock down (and/or an intervention) in an animal model, such as the atherosclerotic mouse. But for SREBPF1 (but especially SREBF2) the necessary experiments have already been performed (example; PMID: 30354203). The authors could mention in the discussion that the effects of the gene in relation to e.g. atherosclerosis are supportive for the observed results.\nResponse:\nWe thank the reviewer for this exceptionally insightful comment. We completely agree that placing our findings in the broader context of general vascular disease mechanisms would significantly strengthen the impact of our work. The connection to atherosclerosis is indeed highly relevant.\nAs suggested, we have now incorporated a discussion on the role of SREBF1 in atherosclerosis and other vascular pathologies to support the generality of our observed mechanism. Specifically, we have added a new paragraph in the Discussion section (page 15, line 34 to page 16, line 16).\nIn this new paragraph, we:\n1. Cite the key paper mentioned by the reviewer (PMID: 30354203) and other relevant literature (PMID: 34094640; 30462530), summarizing the established role of SREBF1/2 and activator SCAP in atherosclerosis.\n2. Explicitly state that these independent findings in a common vascular disease strongly support our results obtained in the microgravity model.\n3. Propose a unifying hypothesis: that SREBF1-mediated lipogenesis and epigenetic remodeling may represent a convergent pathway through which diverse stressors drive VSMC dysfunction.\n4. Conclude that our findings, therefore, not only reveal a mechanism for spaceflight-associated vascular remodeling but also provide novel insights into the fundamental mechanisms underlying vascular aging and disease.\nWe believe this addition has successfully integrated the \"two thoughts\" into a cohesive and more powerful narrative, greatly enhancing the conceptual advance of our manuscript. We are truly grateful for this excellent suggestion.\nReviewer #3 (Remarks to the Author):\nIn the revised manuscript, the authors have addressed all my concerns. This manuscript is ready for publication.\nResponse:\nWe thank the reviewer for their supportive feedback and confirmation that the manuscript is suitable for publication.\nReviewer #4 (Remarks to the Author):\nI 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.\nResponse:\nWe thank the reviewer for their supportive feedback and confirmation that the manuscript is suitable for publication.\nReviewer #5 (Remarks to the Author):\nThank you for taking the time to fully revise the manuscript. I appreciate the authors' efforts in addressing the concerns I raised in my initial review. The revisions have adequately clarified the key issues, and I find the manuscript substantially improved.\nThat said, I would like to emphasize that manuscripts of this complexity and technical depth require careful drafting and thorough internal revision prior to submission. A more rigorous pre-submission review by the authors could have avoided some of the initial issues (like typos and ambiguous acronyms) and facilitated a smoother review process.\nOverall, I am satisfied with the current version and have no further major concerns.\nResponse:\nWe sincerely thank the reviewer for their thoughtful feedback and acknowledge their important point regarding pre-submission preparation. We fully agree that more rigorous internal revision would have streamlined the process, and we have now implemented a stricter pre-submission checklist within our team. Our gratitude for the reviewer's constructive guidance throughout the revision, which has genuinely strengthened the manuscript."}]

Decision letter