Endothelial AGO1 Drives Vascular Inflammation and Atherosclerosis via a Non-Canonical Nuclear Mechanism

Endothelial AGO1 Drives Vascular Inflammation and Atherosclerosis via a Non-Canonical Nuclear Mechanism | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var 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Piotr Swiderski , Kuei-Chun Wang , View ORCID Profile Marcin Kortylewski , Norbert Pardi , View ORCID Profile Lu Wei , Wendong Huang , View ORCID Profile Zhen Bouman Chen doi: https://doi.org/10.1101/2025.05.01.651783 Xuejing Liu 1 Department of Diabetes Complications and Metabolism, Arthur Riggs Diabetes and Metabolism Research Institute, Beckman Research Institute , City of Hope, Duarte, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: zhenchen{at}coh.org xueliu{at}coh.org Dongqiang Yuan 1 Department of Diabetes Complications and Metabolism, Arthur Riggs Diabetes and Metabolism Research Institute, Beckman Research Institute , City of Hope, Duarte, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yingjun Luo 1 Department of Diabetes Complications and Metabolism, Arthur Riggs Diabetes and Metabolism Research Institute, Beckman Research Institute , City of Hope, Duarte, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Xiaofang Tang 1 Department of Diabetes Complications and Metabolism, Arthur Riggs Diabetes and Metabolism Research Institute, Beckman Research Institute , City of Hope, Duarte, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Alonso Tapia 1 Department of Diabetes Complications and Metabolism, Arthur Riggs Diabetes and Metabolism Research Institute, Beckman Research Institute , City of Hope, Duarte, CA, USA 2 Irell and Manella Graduate School of Biological Sciences , City of Hope, Duarte, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Naseeb Kaur Malhi 1 Department of Diabetes Complications and Metabolism, Arthur Riggs Diabetes and Metabolism Research Institute, Beckman Research Institute , City of Hope, Duarte, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Naseeb Kaur Malhi Rahuljeet Singh Chadha 3 Division of Chemistry and Chemical Engineering, California Institute of Technology , Pasadena, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sachchidanand Tiwari 4 Department of Medicine, Perelman School of Medicine, University of Pennsylvania , Philadelphia, PA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Piotr Swiderski 5 DNA/RNA Synthesis Laboratory, Beckman Research Institute , City of Hope, Duarte, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kuei-Chun Wang 6 School of Biological and Health Systems Engineering, Arizona State University , Tempe, AZ, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Marcin Kortylewski 7 Department of Immuno-Oncology, Beckman Research Institute , City of Hope, Duarte, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Marcin Kortylewski Norbert Pardi 4 Department of Medicine, Perelman School of Medicine, University of Pennsylvania , Philadelphia, PA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Lu Wei 3 Division of Chemistry and Chemical Engineering, California Institute of Technology , Pasadena, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Lu Wei Wendong Huang 1 Department of Diabetes Complications and Metabolism, Arthur Riggs Diabetes and Metabolism Research Institute, Beckman Research Institute , City of Hope, Duarte, CA, USA 2 Irell and Manella Graduate School of Biological Sciences , City of Hope, Duarte, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Zhen Bouman Chen 1 Department of Diabetes Complications and Metabolism, Arthur Riggs Diabetes and Metabolism Research Institute, Beckman Research Institute , City of Hope, Duarte, CA, USA 2 Irell and Manella Graduate School of Biological Sciences , City of Hope, Duarte, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Zhen Bouman Chen For correspondence: zhenchen{at}coh.org xueliu{at}coh.org Abstract Full Text Info/History Metrics Preview PDF ABSTRACT BACKGROUND Endothelial cell (EC) dysfunction is both a cause and consequence of vascular inflammation and lipid dysregulation in atherosclerosis, yet the molecular drivers linking EC dysfunction to systemic metabolic derangements remain incompletely understood. We previously identified Argonaute 1 (AGO1)—a canonical component of the RNA-induced silencing complex—as a regulator of EC function in angiogenesis and metabolism. In this study, we uncover a previously unrecognized, non-canonical role of nuclear AGO1 in ECs as a transcriptional coactivator of NF-κB, and demonstrate that EC-specific AGO1 inhibition simultaneously improves lipid metabolism, liver function, and vascular inflammation, thereby attenuating atherosclerosis. METHODS EC-conditional AGO1 knockout (EC-AGO1-KO) and wildtype mice were subjected to pro-atherosclerotic models induced by AAV9-PCSK9 and a western diet, or carotid artery ligation. Metabolic and vascular phenotyping and gene expression analyses were performed. In human liver sinusoidal ECs (HLSECs) and human aortic ECs (HAECs), AGO1 was knocked down using antisense oligos (ASO), followed by assays for inflammatory responses (qPCR, RNA-seq, ELISA, and monocyte adhesion). Mechanistic studies included Cut&Tag sequencing, and chromatin immunoprecipitation assays, and EC-hepatocyte co-cultures. Therapeutic effect of AGO1 inhibition was assessed using lipid nanoparticle (LNP)-delivered ASO in mice. RESULTS EC-AGO1-KO mice exhibited significantly improved plasma lipid profiles, reduced hepatic steatosis, inflammation, and fibrosis, and decreased aortic atherosclerotic burden. AGO1 knockdown in ECs dampened inflammatory responses and monocyte recruitment and enhanced hepatocyte lipid metabolism via paracrine signaling. Mechanistically, nuclear AGO1 interacted with NF-κB p65 to enhance transcription of pro-inflammatory genes including ICAM1 , THBS1 . LNP-delivered AGO1-ASO improved hyperlipidemia, liver function, and atherosclerosis without evident hepatotoxicity. CONCLUSIONS Endothelial AGO1 promotes vascular inflammation and liver dysfunction through a non-canonical role as an NF-κB coactivator. Its inhibition provides dual benefits—ameliorating lipid dysregulation and suppressing vascular inflammation—highlighting EC-AGO1 as a promising therapeutic target for atherosclerosis and cardiometabolic diseases. INTRODUCTION Atherosclerosis (AS), a major cause of cardiovascular disease, arises from hypercholesterolemia due to liver dysfunction and from chronic vascular inflammation. 1 One of the earliest events that contributes to AS is the dysfunction of endothelial cells (EC), which forms the inner lining of the blood vascular wall. 2 In the classic model of AS, cholesterol-loaded low-density lipoproteins (LDLs) become oxidized in the arterial intima and increase expression of adhesion molecules such as intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1). The upregulation of these adhesion molecules promotes the recruitment and residence of leukocytes, e.g., monocytes, which further differentiate into macrophages, propagating the pro-inflammatory cascade. 3 The crucial role of EC dysfunction in AS is well recognized and substantiated by a large body of literature. Various mouse models with EC-conditional deletion or overexpression of genes encoding adhesion molecules, membrane receptors, adaptor proteins, and transcription factors (TFs) have been characterized for vascular inflammation and remodeling and altered atherosclerotic burden. 3 The underlying mechanisms include, but are not limited to, the regulation of oxidative stress, 4 , 5 nitric oxide (NO) bioavailability, 6 leukocyte adhesion, 7 endothelial permeability, 8 , 9 and endothelial-to-mesenchymal transition. 10 Moreover, targeting EC dysfunction using pharmacological approaches such as nanoparticles or EC-directed peptides can attenuate the AS lesions in mice. 11 – 14 These findings underscore the critical role of endothelial function in AS and suggest that targeting ECs may provide therapies to treat AS. Few studies have addressed the role of EC dysfunction in the development of hyperlipidemia in the context of AS. Mice with EC-specific gene perturbation typically develop exacerbated or attenuated AS lesions without changes in their lipid profile. Given the large surface area of ECs in the body and the liver as a highly vascularized organ where liver sinusoidal ECs interact closely with hepatocytes through cytokine, angiocrine, and metabolic signals, 15 – 18 EC dysfunction may contribute to dysregulated liver function in AS. In support of this view, EC-specific loss of VCAM1 attenuates hepatic inflammation and fibrosis without changing lipid metabolism in mice fed a choline-deficient high-fat diet to mimic metabolic dysfunction-associated steatohepatitis (MASH). 19 Overexpression of ATP binding cassette subfamily A member 1 (ABCA1), a key membrane transporter for cholesterol efflux in ECs, elevates plasma high-density lipoprotein (HDL) and mitigates AS induced by a high-fat, high-cholesterol diet (HFHCD). 20 However, whether inhibiting an endogenous gene in ECs can impact liver function, lipid profile, and the vascular inflammation in the context of AS has not been demonstrated. In mammalian cells, Argonaute 1 (AGO1) has been characterized mostly as a key protein of the RNA-induced silencing complex (RISC), 21 with less characterized nuclear function to regulate gene transcription. 22 Unlike its homologous AGO2 protein, AGO1 does not possess endonuclease activity to cleave mRNAs. While global AGO2-knockout (KO) mice display severe developmental abnormalities and are embryonically lethal, 23 AGO1-KO mice are viable. 24 We found in ECs that hypoxia downregulates AGO1 to promote angiogenesis, in part through microRNA (miR)-mediated post-transcriptional regulation of pro-angiogenic vascular endothelial growth factor A (VEGFA) and angiostatic thrombospondin (TSP1, encoded by THBS1 ). 25 , 26 Furthermore, mice with EC-AGO1 deficiency develop an anti-obesity phenotype with increased fat browning and insulin sensitivity. 26 These findings and recent reports showing an active role of EC function in modulating metabolic function, 19 , 27 – 29 support the idea that ameliorating EC dysfunction may improve metabolic state. 27 , 30 Thus, we investigated the effects of inhibition of EC-AGO1 on liver dysfunction and vascular inflammation, as well as the underlying molecular and cellular mechanisms. In this study, EC-AGO1-KO mice were subjected to an atherogenic regimen and their lipid profiles, liver function, and atherosclerotic lesions were characterized. The effect of AGO1 inhibition in human liver sinusoidal ECs (HLSEC) and human aortic ECs (HAEC), two EC types affected in hypercholesterolemia and AS, was determined. EC-AGO1 inhibition in the liver and in the aortic wall in vivo and in vitro resulted in an anti-atherosclerotic phenotype. Mechanistically, we identified a non-canonical nuclear role of AGO1, by interacting with NF-κB to induce the expression of pro-inflammatory genes in ECs. Finally, lipid nanoparticle (LNP) inhibition of AGO1 attenuated hypercholesterolemia and atherogenesis. Collectively, our findings highlight EC-AGO1 as a critical regulator in AS through metabolic and vascular modulations. Targeting EC-AGO1 may exert dual benefits in metabolic and vascular regulation to treat AS. RESULTS EC-AGO1-KO mice show improved lipid profile and liver function under metabolic stress We previously established a mouse line by crossing Cdh5-cre and AGO1-floxed mice to study the effect of EC-AGO1-KO on obesity and adipose tissue function. 26 When subjected to an obesogenic high-fat, high-sucrose (HFHS) diet, along with the previously observed anti-obesity phenotype, 26 EC-AGO1-KO mice exhibited lower levels of total cholesterol (TC) and low-density lipoprotein/very-low-density lipoprotein (LDL/VLDL) cholesterol, and higher levels of HDL cholesterol levels, with comparable triglyceride (TG) levels (Figure S1). Given the focus on AS in this study, we validated that AGO1 was reduced in ECs isolated from the liver and aorta, but not in monocytes from the EC-AGO1-KO mice compared to wildtype (WT) littermates ( Figure 1A , Figure S2A). Of note, the expression of AGO2, another AGO family member more intensively studied, was not affected by AGO1-KO in ECs (Figure S2B). Download figure Open in new tab Figure 1. EC-AGO1-KO mice show improved lipid profile and liver function in an AAV9-PCSK9-induced atherosclerosis model. A, mRNA levels of AGO1 in ECs isolated from the liver and aorta quantified by qPCR and normalized to 36B4 (n=3 per group). B-J , Male EC-AGO1-KO mice and WT littermates (12-week-old) were injected with AAV9-CTRL/PCSK9 and fed a high fat high cholesterol diet (HFHCD) for 16 weeks (n=5-7 per group). B , Experimental scheme. C , Representative plasma samples from three groups of mice described in ( B ). D-G , Levels of triglyceride (TG), total cholesterol (TC), LDL/VLDL, and HDL in the plasma after 4 hours fasting. H , I , Plasma ALT and AST levels. J , Representative images of HE, lipid channel (-CH 2 , 2845 cm -1 ) from SRS imaging, F4/80, and Masson Trichrome staining of the liver. Data are presented as mean±SEM in ( A and D-I ). *p<0.05, **p<0.01, ***p<0.001 between the indicated groups based on Student’s t-test ( A ) and one-way ANOVA ( D-I ). Next, we subjected the WT and KO mice to an atherogenic model induced by AAV9-PCSK9 ( Figure 1B ), which elevates plasma LDL-cholesterol levels by promoting the degradation of hepatic LDL receptors (LDLR). 31 When combined with a HFHCD, AAV9-PCSK9 induces a robust hyperlipidemia and AS phenotype. 32 AGO1 may be involved in the host response to viral infection. 24 However, the WT and KO have comparable uptake of AAV9-tdTomato (Figure S3). Following AAV9-PCSK9 administration, PCSK9 expression increased in a dose-dependent fashion compared to the control AAV, reaching ∼20-fold induction in mice given 10 11 vector genomes (vg) (Figure S4), which was employed in subsequent experiments. Compared to WT mice given the control AAV (AAV-CTRL) and HFHCD, WT mice receiving AAV9-PCSK9 and HFHCD developed pronounced hyperlipidemia with milky-white plasma, elevated plasma TG, TC, and LDL/VLDL and diminished HDL ratio. In contrast, the EC-AGO1-KO mice subjected to the same regimen showed much less hyperlipidemia, hypercholesterolemia, and chylomicronemia ( Figure 1C-G ) without significant differences in body weight (Figure S5). In WT mice, the atherogenic regimen increased the plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which were almost reversed in the KO mice ( Figure 1H , I). Histological analysis of livers from WT mice showed severe steatosis, and this was drastically reduced in livers from the EC-AGO1-KO. Specifically, the KO livers had lower lipid deposition (revealed by HE staining and stimulated Raman scattering [SRS] imaging of lipids), macrophage infiltration (stained by F4/80), and fibrosis (visualized by Masson’s Trichrome staining) ( Figure 1J ). Of note, chow-fed mice receiving AAV-PCSK9 did not have a significant difference in body and liver weight, or in the plasma levels of TG, TC, and LDL/VLDL levels, although the HDL levels were higher, and the ALT and AST were lower in EC-AGO1-KO mice (Figure S6). In summary, after exposure to an atherogenic diet, EC-AGO1-KO mice exhibited significantly improved plasma lipid profile and reduced hepatic steatosis, inflammation, and fibrosis compared to WT mice. EC-AGO1-KO mice show attenuated atherosclerotic lesions As expected, en face Oil Red O (ORO) staining revealed no apparent plaque in the aortas of the WT mice receiving AAV-CTRL despite the HFHCD. This was in stark contrast to the extensive plaque found in the aortic arch, thoracic aorta, abdominal aorta, and aortic roots from WT mice receiving AAV9-PCSK9 and HFHCD. However, the same regimen induced significantly less plaque burden in all vascular regions examined in the EC-AGO1-KO mice ( Figure 2A-D , Figure S7). Download figure Open in new tab Figure 2. EC-AGO1-KO mice show attenuated aortic atherosclerotic lesion. The same mice described in Figure 1 were analyzed. A, B , En face Oil red O (ORO) staining of the aortic tree and root. C, D , Quantification of atherosclerotic lesion areas based on ORO staining in the entire aorta (in C ) and the aortic root (in D ). E, IHC for F4/80, α-SMA, and Masson’s trichrome staining in the aortic root. F, Raman SRS imaging of the aortic root. The first row represents a mosaic of the protein channel (CH 3 , 2845 cm -1 ), followed by a zoomed-in image of the lipid channel (CH 2 , 2845 cm -1 ), a corresponding lipid-to-protein ratiometric image, and an image targeted at the phosphate channel (PO 4 3- , 960 cm -1 ). Scale bar = 100 mm. Representative images from 5-7 mice/group. Data are presented as mean±SEM ( C, D ). ***p<0.001 between indicated groups based on one-way ANOVA. Histological analyses showed that the AAV9-PCSK9-mediated increase in macrophage infiltration (stained by F4/80) and decrease in smooth muscle cells (SMC) content (stained by α-SMA) and collagen (stained by Masson’s trichrome) were strongly attenuated in the KO mice ( Figure 2E ). To further characterize the biochemical composition of the AS lesions, we utilized label-free SRS microscopy which allows non-invasive and high-resolution mapping of metabolites by detecting their vibrational signatures in tissues. 33 – 35 In addition to detecting increased lipids in the AS plaques from AAV9-PCSK9 treated WT mice, SRS microscopy revealed increased phosphate (PO 4 3− , ν 1 stretching) signals corresponding to the microcalcifications often noted in unstable AS plaques. 36 These metabolite changes were much less in KO mice subjected to the same atherogenic regimen ( Figure 2F ). Together with lipid profile and liver data, these results demonstrate anti-atherosclerotic tendencies from EC-AGO1-KO. Inhibition of AGO1 in ECs exerts an anti-inflammatory effect Inflammation is a link between EC dysfunction and AS 3 . We tested whether the inhibition of AGO1 in ECs exerts an anti-inflammatory effect to lessen AS. To this end, we designed locked nucleic acid-GapmeR-based antisense oligos (ASOs) that target both human and mouse AGO1 (Table S1). Among three ASOs, ASO-112 (termed AGO1-ASO) that showed the strongest and most consistent knockdown (KD) (Figure S8) was employed. In HLSECs and HAECs, AGO1-KD attenuated induction of pro-inflammatory genes including ICAM1, VCAM1, and CCL2 (encoding monocyte chemoattractant protein/MCP1) by oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (oxPAPC), a major component of minimally modified LDL shown to promote EC dysfunction 37 ( Figure 3A-D , F-I). Additionally, AGO1-KD also suppressed THBS1 ( Figure 3E , J) which can also promote EC dysfunction and vascular inflammation. 38 Download figure Open in new tab Figure 3. Inhibition of AGO1 in ECs exerts anti-inflammatory effect. A-L, Human liver sinusoidal ECs (HLSECs) (in A-E) and human aortic ECs (HAECs) (in F-L ) were transfected with scramble (Scr) or AGO1-ASO (20 nM), before treated with oxPAPC (40 µg/mL) or a vehicle control for 4 hours. A-J, mRNA levels of genes as indicated were quantified by qPCR, with b-actin as an internal control. K, L, Attachment of fluorescently labeled THP-1 monocytes to HAECs were imaged and quantified. Shown are relative fold change, with Scr-vehicle control set as 1. M, N, qPCR quantification of mRNA levels of genes involved in inflammation in the liver and aorta of mice described in Fig. 1 . O-Q, Partial carotid artery ligation (PCL, illustrated in O ) was performed on 12-week-old male EC-AGO1-KO mice and WT littermates fed a chow diet. The ligated left carotid artery (LCA) and the non-ligated right carotid artery (RCA) were harvested 1-week post-ligation. mRNA levels of Ccl2 (in P ) and Thbs1 (in Q ) were quantified by qPCR and normalize to 36B4. Scale bar = 200 µm. Data represents mean±SEM of three ( A-J ) and six ( K, L ) independent experiments and n=6-7/group ( M, N ) and n=3/group ( O-Q ) of mice. *p<0.05, **p<0.01, and ***p<0.001 based on two-way ANOVA ( A-L ) and Student’s t-test ( M, N and P, Q ). In line with the effect of AGO1-KD on gene expression, oxPAPC-induced monocyte adhesion to HAECs was abolished by AGO1-KD ( Figure 3K , L). This anti-inflammatory effect of AGO1-KD was also observed in human umbilical vein endothelial cells (HUVECs) exposed to high glucose and TNF-α (HT), a condition known to drive EC dysfunction and commonly associated with metabolic disorders, including diabetes, 39 , 40 a major risk factor for AS (Figure S9). Consistent with the in vitro data, the expression levels of the inflammation markers, i.e., Icam1 , Vcam1 , Ccl2, and Thbs1, were also decreased in the liver and aorta of EC-AGO1-KO mice subjected to the AS regimen, as compared to the WT controls ( Figure 3M , N). The decreased hyperlipidemia noted in the EC-AGO1-KO mice ( Figure 1 ) might have also accounted for less AS in these mice. To test if EC-AGO1-KO directly affects vascular inflammation in vivo , we partially occluded the left carotid artery (LCA) in mice kept at baseline. The resultant disturbed blood flow causes EC dysfunction and vascular inflammation without altering the plasma lipid profile 41 ( Figure 3O ). As expected, the inflammatory markers Ccl2 and Thbs1 were strongly induced in the ligated LCA as compared to the non-ligated right carotid artery (RCA) in the WT mice. However, in the EC-AGO1-KO mice, partial carotid artery ligation (PCL) had minimal effect on gene expression ( Figure 3P , Q). Collectively, these data indicate that AGO1 suppression in ECs confers an anti-inflammatory effect in the liver and arteries. Inhibition of AGO1 in ECs affects hepatocyte gene expression To assess the effect of AGO1 inhibition on the EC transcriptome, we performed bulk RNA-seq of HAECs. Compared to the scramble control (Scr), AGO1-ASO resulted in a total of 195 differentially expressed genes (DEGs) (including 93 up- and 102 down-regulated) in ECs-treated with oxPAPC. The identified DEGs are enriched for immune and inflammatory response pathways, e.g., TNF-α signaling, NOD-like receptor signaling, and NF-κB signaling ( Figure 4A ). A panel of oxPAPC-induced inflammatory genes, including adhesion molecules ( ICAM1 and VCAM1 ), chemokine ligands (i.e., C-C motif chemokine ligands [CCLs] and C-X-C motif chemokine ligands [CXCLs]), and interleukins (ILs), were suppressed by AGO1-KD ( Figure 4B ). In parallel, we analyzed the cytokine production from ECs using the Luminex assay ( Figure 4C ). At baseline, AGO1-KD did not significantly affect the levels of most of the cytokines assayed. However, in ECs treated with oxPAPC, AGO1-KD significantly reduced the levels of several pro-inflammatory cytokines including IL-7, IL-8, IL-33, and IFN-α ( Figure 4D-G ). Download figure Open in new tab Figure 4. Inhibition of AGO1 in ECs alters gene expression in the hepatocytes and liver. A, B, Bulk RNA-seq of HAECs treated with oxPAPC and transfected with ASO (same as in Figure 3 ). A, Enriched pathways based on KEGG as the effect of AGO1-KD in oxPAPC-treated HAECs. B , Heat map showing induction of genes involved in the cytokine and immune response pathways in HAECs by oxPAPC and downregulation by AGO1-KD. C-J , HUVECs were transfected with ASO for experiments illustrated in ( C ). In ( D-G ), transfected ECs were treated with oxPAPC or vehicle control for 4 hours. The media was collected for Luminex ELISA. In ( H-J) , transfected ECs were treated with TNF-α (5 ng/mL) and co-cultured with HepG2 cells in the Transwell for 24 hours. The mRNA levels of various genes in HepG2 cells were quantified by qPCR. K-N , Liver samples from mice described in Figure 1 were processed for qPCR (in K, L ) and Western blotting (in M, N ). Representative image ( M ) and quantification ( N ) of Western blotting for p-ACC1 and p-AMPK. Data are presented as mean±SEM. *p<0.05, **p<0.01, and ***p<0.001 based on two-way ANOVA ( D-G ), Student’s t-test ( H-J, N ) and one-way ANOVA ( K, L ). Paracrine signaling between ECs and hepatocytes has been reported. 15 , 17 , 18 , 42 To this end, we set up EC-hepatocyte co-cultures and tested the impact of AGO1 inhibition on hepatocytes. ECs with or without AGO1-KD were treated by TNF-α and then co-cultured with HepG2 cells on the opposite side of the Transwell chamber ( Figure 4C ). As a result of AGO1-KD in ECs, inflammatory marker genes i.e., ICAM1 , VCAM1 , CCL2 and THBS1 were downregulated in HepG2 cells. Moreover, several genes involved in lipid metabolism, i.e., CD36 for lipid uptake, SREBP1 and FASN for de novo lipogenesis (DNL), HMGCR and LDLR for cholesterol synthesis and uptake, LXRα for cholesterol degradation were upregulated in HepG2 cells ( Figure 4H-J ). Of note, AGO1 expression in HepG2 cells were unchanged ( Figure 4H ). As a control, direct AGO1-KD in HepG2 cells did not cause any notable changes in the expression of these genes (Figure S10). These data suggest that EC with AGO1 inhibition confers anti-inflammatory and metabolic protective effects on hepatocytes. Similar to cell studies, upregulation of genes involved in lipid metabolism was found in the livers of the KO mice exposed to the AAV-PCSK9-induced atherogenic regimen, as compared to the WT littermates ( Figure 4K , L). Of note, these differences were absent in mice at baseline (Figure S11). At the protein level, markers of metabolic homeostasis and liver function including phospho-Acetyl-CoA Carboxylase (p-ACC) and phospho-adenosine monophosphate-activated protein kinase (p-AMPK), were significantly elevated in EC-AGO1-KO mouse livers, indicating an improved metabolic function ( Figure 4M , N). Collectively, these findings suggest that the inhibition of AGO1 in ECs improves hepatocyte and liver metabolic function. AGO1 localizes to the nucleus and interacts with NF-κB to drive pro-inflammatory gene expression in ECs The strong anti-inflammatory effect and the decreased mRNA levels of pro-inflammatory genes due to AGO1 KO/KD in ECs suggest a positive regulation of inflammation by AGO1, likely through transcriptional regulation. In cancer cells, AGO1 was shown to bind to promoters and interact with RNA polymerase II (RNAP II) and TFs such as estrogen receptor (ER) to facilitate gene activation. 43 , 44 To probe if such a mechanism exists in non-cancerous cells like ECs, we first performed immunofluorescent (IF) staining of AGO1. We observed AGO1 in the nucleus and cytoplasm in ECs at baseline and under inflammatory stress without significant difference in either protein levels or subcellular localizations ( Figure 5A-D ). This was consistent with Western blotting of AGO1 in subcellular fractionations from ECs ( Figure 5E ). To assess the function of nuclear AGO1 in ECs, we performed Cut&Tag-sequencing (Cut&Tag-seq) with an AGO1 antibody, with IgG as an isotype control. Analysis of the Cut&Tag-seq data revealed strong binding between AGO1 and chromatin, which was increased by oxPAPC but decreased by AGO1-KD in ECs ( Figure 5F ). Specifically, in oxPAPC-treated ECs, gene promoters accounted for 24% of AGO1-bound genomic regions, which was reduced to 19% by AGO1-KD ( Figure 5G ). Download figure Open in new tab Figure 5. AGO1 interacts with NF-kB to regulate the pro-inflammatory gene expression. A-E , HUVECs and HAECs were treated by 25 mM mannitol as normal glucose and osmolarity control (NM) or 25 mM D-glucose and 5 ng/mL TNF-α (HT) ( A-D ), or 5 ng/mL TNF-α only ( E ). Confocal imaging of AGO1 immunofluorescent (IF) staining was performed with DAPI counterstain ( A, B ) and the subcellular localization was quantified ( C, D ). Scale bar =20 mm. e , Representative Western blot of AGO1 in subcellular fractions from β-tubulin and Lamin-B1 were used as cytoplasmic and nuclear markers. WL=whole cell lysates, Nuc=nuclear extracts, and Cyto=cytoplasmic extracts. F, G , HUVECs transfected by ASO and treated with or without oxPAPC underwent Cut&Tag-seq with AGO1 antibody, with IgG as an isotype control. F , AGO1 occupancy in the chromatin, with enriched peaks near transcription start sites (TSS). G , Distribution of AGO1-binding peaks across the promoter and other genomic regions in oxPAPC-treated ECs without or with AGO1-KD. H , Enriched pathways in the 86 genes that are regulated by AGO1 and show promoter binding by AGO1. I , Gene tracks showing the binding of AGO1 in the genomic loci encoding indicated marker genes. J , qPCR of AGO1 binding at the promoter regions of THBS1 and ICAM1 performed with Cut&Tag samples from ( F, G ). K , Top 5 TFs (including NF-kB RELA) whose TFBS were enriched in AGO1-bound DNA sequences identified by Cut&Tag. L , AGO1 protein-protein interaction network constructed based on yeast-two hybridization. M , HUVECs were treated with or without oxPAPC. Co-IP of AGO1 and NF-κB p65, with IgG as an isotype control. N , Representative confocal microscopy images of AGO1 (red) and p65 (green) in HAECs treated with HT. The arrow in each merged image indicates the plane for generating line profiles and calculation of Pearson’s correlation coefficient (r). The co-localization of AGO1 and p65 in the nucleus were quantified in 20 cells per group. Scale bar = 20 mm. O , HAECs were treated with HT without or with AGO1-KD. ChIP was performed with p65 antibody and the association of p65 with THBS1 and ICAM promoters was detected using qPCR. *p<0.05 and ***p<0.001 based on Student’s t-test ( N, O ). Cross-referencing the RNA-seq ( Figure 4A , B) and Cut&Tag-seq data, we found that out of 102 genes downregulated by AGO1-KD in oxPAPC-treated ECs, 86 showed association with AGO1 in their promoter regions. Sixty of these genes are involved in the EC immune or inflammatory responses ( Figure 5H ), including THBS1 and ICAM1 ( Figure 5I ) . As validation, qPCR revealed that AGO1 indeed binds to the peak regions in the promoters of THBS1 and ICAM1 as identified by Cut&Tag-seq and AGO1-KD decreased these interactions oxPAPC-treated ECs ( Figure 5J ). In breast cancer cells, AGO1 was found to act as a transcriptional co-activator of ER alpha. 44 To identify potential TFs involved in AGO1-regulated inflammatory gene program in ECs, we analyzed TF binding sites (TFBS) in the promoter regions of 86 genes that are both AGO1-bound and AGO1-positively regulated. TRANSFAC identified NF-κB (subunit P65, aka RELA), as one of the top candidates ( Figure 5K ). Out of the 86 genes identified, 17 have been reported to be regulated by NF-κB with putative p65 TFBS 45 , 46 (Table S2). Additionally, NF-κB signaling was among the top enriched pathways in DEGs caused by AGO1-KD ( Figure 4A ). Search using databases for protein-protein interactions (PINA https://omics.bjcancer.org/pina and INTACT https://www.ebi.ac.uk/intact ) revealed that AGO1-RELA interaction has been experimentally verified by using a yeast-two hybrid system 47 ( Figure 5L ). Co-immunoprecipitation (co-IP) validated that AGO1 can indeed interact with RELA/p65 in EC ( Figure 5M ). Furthermore, Co-IF detected AGO1 and p65 co-localization in ECs, especially under the pro-inflammatory treatment ( Figure 5N , Figure S12). To examine the potential role of AGO1 in the NF-κB-promoted inflammatory gene expression, we performed ChIP with p65 antibody in HT-treated HAECs. qPCR revealed that p65 binding to the promoter regions of ICAM1 and THBS1 was decreased by AGO1-KD ( Figure 5O ). Together, these results suggest that AGO1 binds to NF-κB to promote transcription of pro-inflammatory genes in ECs. Lipid nanoparticle (LNP) delivery of AGO1 ASO confers anti-hyperlipidemic and anti-atherosclerotic effects Given that EC-AGO1-KO mice showed anti-hyperlipidemic and anti-atherosclerotic tendencies, we evaluated the therapeutic potential of AGO1-ASO to treat AS. We confirmed the efficacy of AGO1-ASO in WT mice. Intravenous injection (i.v.) of AGO1-ASO (10 mg/kg body weight) decreased Ago1 mRNA levels in the liver of WT mice by more than 50% (Figure S13A). When encapsulated in LNP, AGO1-ASO (1 mg/kg body weight, i.e., 1/10 th of the dose without LNP) reduced AGO1 mRNA by 75% (Figure S13B) and provided a dose for further study. WT mice were given AAV9-PCSK9 and HFHCD. Two weeks later, mice received weekly i.v. injections of LNP-AGO1-ASO or LNP-Scr for 4 weeks ( Figure 6A ). LNP-AGO1-ASO significantly reduced the body weight compared to LNP-Scr-treated mice ( Figure 6B ). LNP-AGO1-ASO-treated mice had almost clear plasma ( Figure 6C ), significantly reduced TG, TC, and LDL/VLDL levels, and increased HDL/TC ratio ( Figure 6D-G ). At the gene expression level, pro-inflammatory ( Icam1, Vcam1, Ccl2, Thbs1 ) and pro-fibrotic ( Acta2, Collagen I, Collagen III ) genes were decreased; several genes involved in lipid metabolism such as Srebp1, Acc1, and Scd1 were increased and Abcg1 was decreased by LNP-AGO1-ASO in the liver ( Figure 6H ). A similar anti-inflammatory effect was also observed in the aorta ( Figure 6I ). Download figure Open in new tab Figure 6. Lipid nanoparticle (LNP) delivery of AGO1 ASO is anti-hyperlipidemic and anti-atherosclerotic. A , Experimental design: male C57BL6 wildtype mice (20-week-old, n=5/group) received AAV9-PCSK9 and were fed a HFHCD. Two weeks later, LNP-ASO was injected through tail vein at 1 mg/kg body weight weekly for 4 consecutive weeks. B , Body weights were measured biweekly. C , Plasma samples were collected from LNP-ASO treated mice after 4-hour of fasting. D-G , Plasma lipid profiles. H, I , qPCR quantification of mRNA levels of various genes in the liver (in H ) and aorta (in I ). J , Representative images of HE, ORO, F4/80 and Masson’s Trichrome staining of the liver. K-N , Representative images of ORO staining of the aortic arch and root and the quantification. O , Schematic summary of the proposed mechanism of AGO1-regulated inflammatory response in ECs in the context of hyperlipidemia and atherosclerosis (left) and the effect of EC-AGO1 inhibition on vascular inflammation, liver dysfunction, and atherosclerosis (right). Data are presented as mean±SEM, *p<0.05, **p<0.01 based on Student’s t-test. LNP-AGO1-ASO-treated mice also had significantly less hepatic lipid accumulation, macrophage infiltration, and fibrosis ( Figure 6J ). Importantly, LNP-AGO1-ASO treatment did not affect liver size, or plasma ALT and AST levels (Figure S14), suggesting it was not liver-toxic. Finally, the AS lesions in the aortic arch and root were substantially attenuated in LNP-AGO1-ASO-treated mice ( Figure 6K-N ). These findings demonstrate that systemic AGO1 inhibition is anti-hyperlipidemic and anti-atherosclerotic. DISCUSSION This study points out a critical role of AGO1 in driving EC dysfunction, vascular inflammation, and lipid dysfunction in the context of AS ( Figure 6O ). EC-AGO1-KO mice exposed to AAV-PCSK9 and an atherogenic diet exhibited an improved lipid profile and liver function, decreased hepatic steatosis, inflammation, and fibrosis, and substantially attenuated AS lesions. Likewise, LNP-AGO1-ASO treated WT mice resisted the metabolic and vascular derangements induced by PCSK9 and an atherogenic diet. Together with our previous findings showing an anti-obesity and anti-insulin resistance phenotype in EC-AGO1-KO mice subjected to a HFHS diet, 26 the pleiotropic effects of EC-AGO1 inhibition underscore the importance of EC dysfunction in both metabolic disorders and vascular diseases. The benefits of EC-AGO1-KO and inhibition, as shown in vitro and in vivo , indicate a consistent anti-inflammatory effect in the liver and in the aortas/arteries. Remarkably, these effects were mostly observed in ECs and animals exposed to metabolic stress, but not at baseline, suggesting a context-dependent aspect to AGO1 KO or inhibition in ECs. Given the strong suppressive effect on hypercholesterolemia in mice with EC-AGO1 inhibition, the reduced AS burden could be due to improved lipid profile, rather than reduced inflammation in the arteries. Independent from hyperlipidemia, we found that EC-AGO1-KO mice subjected to PCL and disturbed flow patterns showed significantly decreased inflammatory gene expression ( Ccl2 and Thbs1 ) in their arterial wall. Thus, EC-AGO1 inhibition likely exerts anti-inflammatory effects in both liver and blood vessels, together contributing to an anti-atherosclerotic phenotype. The anti-inflammatory effect is a reasonable explanation for the hepatic protective effect of EC-AGO1 inhibition. Indeed, in oxPAPC-treated ECs, EC-AGO1-KD decreased expression of adhesion molecules (ICAM1 and VCAM1) and inflammatory agents (THBS1, IL1A, and IL1B) and chemokines (CCL2, CXCL1, and CXCL2). These data are consistent with the decreased monocyte adhesion to ECs with AGO1-KD and reduced macrophage infiltration into the liver of EC-AGO1-KO mice. These findings align with the reduced hepatic inflammation and fibrosis in the EC-VCAM1-KO mice subjected to a MASH model, although the lipid metabolism was unaltered, 19 alluding to a direct role of ECs that impacts hepatocyte function. For example, LSECs secret WNT2 to control cholesterol uptake and bile acid conjugation in hepatocytes. 15 Semaphorin 3a (Sema 3a), a secreted glycoprotein from ECs regulates the number of fenestrae of LSECs and EC-Sema3a-KO attenuated fatty liver in mice. 48 In our study, AGO1-KD decreased the production of IL-7, IL-8, IL-33, and IFN-α, all of which can promote hepatocyte dysfunction. 49 – 52 Furthermore, the coculture experiments demonstrated that ECs with AGO1-KD promoted a gene profile that could enhance fatty acid and cholesterol metabolism. How ECs modulate hepatocyte function warrants further investigation. AGO1 has been extensively characterized in the context of miR-mediated gene silencing. We previously demonstrated that AGO1 regulates the THBS1 expression through miR-mediated post-transcriptional silencing, which may contribute to EC dysfunction, obesity, and insulin resistance. 26 As expected, THBS1 expression was consistently decreased by AGO1-KD/KO in oxPAPC-treated ECs and in the mouse liver and the aortic tree. Given the reported role of TSP1 in EC dysfunction, inflammation, and fibrosis in the context of MASH and AS, 53 , 54 inhibition of THBS1 probably contributes to the beneficial effects of EC-AGO1 inhibition. Aside from this mechanism and the canonical role of AGO1 in RISC, we identified a non-canonical mechanism by which AGO1 interacts with NF-κB (a master regulator of inflammation 55 ) and genomic loci encoding key inflammatory genes in the nucleus of ECs. Of note, p65/RELA expression is not affected by AGO1-KD. Moreover, the ChIP-qPCR data suggest that AGO1 is necessary for p65 binding to the promoters of its transcriptional target. A plausible model for the nuclear AGO1-mediated mechanism is that upon inflammatory stimuli, nuclear translocated p65 interacts with AGO1, which acts as a co-activator for NF-κB-transactivated gene expression. This is reminiscent of the finding that AGO1 was necessary for ER binding to the promoters of its target genes in breast cancer cells upon estradiol treatment 44 and in line with other reports on the function of AGO1 in transcriptional regulation. 43 , 56 , 57 Given the case of THBS1 , such nuclear action of AGO1 is likely in synergy with its cytoplasmic function in mediating RISC, collectively contributing to EC dysfunction under pro-inflammatory conditions. In a treatment study, WT mice challenged with the atherogenic regimen but administered LNP-AGO1-ASO had improved lipid profiles and attenuated AS without evidence of liver toxicity. The systemic administration of LNP-AGO1-ASO is likely to inhibit AGO1 in many cell types. Suggesting some cell specificity to the therapeutic effect, AGO1-KD in the hepatocytes did not lead to similar changes as those noted in EC-AGO1-KD. Moreover, hepatocyte-specific AGO1-KO mice did not show significant difference in liver function or whole-body metabolism either at baseline or under a high-fat diet. 58 Thus, the consistent phenotypes of AGO1-ASO-treated mice with those of EC-AGO1-KO mice, including gene expression, histology, lipid profile, and AS burden indicate that ECs are an important effector contributing to the anti-hyperlipidemic and anti-atherosclerotic effects of AGO1-ASO. Our data and other reports, 26 , 27 , 59 , 60 foster the view that EC-based therapeutics may concurrently improve metabolic and cardiovascular outcomes. 30 RNA-based therapeutics are in development for human diseases. 61 , 62 Application of this technology to AGO1 suggests a means of ameliorating EC dysfunction and vascular inflammation in the liver and arterial wall, conferring a protective effect against hypercholesterolemia and its associated AS. These findings position endothelial AGO1 as a promising therapeutic target, highlighting the potential of RNA-based strategies to simultaneously combat hyperlipidemia, vascular inflammation, and atherosclerosis. Limitations of the Study We acknowledge several limitations of our study. First, the use of Cdh5-cre to cause EC-AGO1-KO may target AGO1 in hematopoietic cells, which can impact the atherogenic process. By analyzing the bone marrow-derived monocytes, we did not observe a significant change in AGO1 expression in the EC-AGO1-KO mice. However, we cannot exclude the possibility that AGO1 may regulate the functions of other hemopoietic cells such as B or T cells that play important roles in AS. Second, our in vivo experiments were predominantly performed with male mice as female mice are generally resistant to hyperlipidemia and AS. 63 , 64 Indeed, the atherogenic regimen used in male mice induced only moderate hyperlipidemia in female mice. Nonetheless, the female EC-AGO1-KO mice showed a trend toward decreased LDL/VLDL levels and the female mice that received AGO1-ASO had a trend toward lower body weight and lower expression of inflammatory markers ( Icam1 and Ccl2 ) in the liver and aorta, compared to controls (Figure S15), suggesting AGO1 may contribute to the metabolic and vascular disease in the female. Finally, to assess the therapeutic potential of AGO1 inhibition, we used LNP delivery, which can also target other cell types beyond ECs. Targeting with nanoparticles and peptides might allow precise drug delivery to ECs only. METHODS Study Approval All animal experiments conducted were approved by the Institutional Animal Care and Use Committees at City of Hope (#17010) and Institutional Biosafety Committee (#16023). Mouse Models EC-AGO1-KO and their WT littermates were generated by crossbreeding VE-Cadherin-Cre (B6.FVB-Tg [Cdh5-cre]7 Mlia/J) and AGO1 flox/flox (Ago1tm1.1Tara/J) mice, both with C57BL6 at City of Hope as described. 26 The WT and KO littermates from the same breeders were housed in the same cages until subjected to metabolic phenotyping and tissue collection. At end points of each experiment, mice were euthanized with CO 2 inhalation. Chow and HFHS diet feeding Mice were fed a chow diet (D12489B, Research Diets Inc, 16.4% kcal protein, 70.8% kcal carbohydrate, 4.6% kcal fat) or an irradiated HFHS (D12266B, Research Diets Inc, 17% kcal protein, 32% kcal fat, 51% kcal carbohydrate) diet for 16 weeks starting at 8 weeks old. AAV9-PCSK9 model Mice were injected in the tail vein with AAV-CTRL or recombinant AAV serotype-9 expressing the PCSK9 mutant under the hepatic control region-apolipoprotein enhancer/alpha1-antitrypsin, a liver-specific promoter (AAV9-HCRApoE/hAAT-D377Y-mPCSK9, abbreviated as AAV-PCSK9) 13 at 1×10 11 vg and fed a chow diet or a HFHCD (Harlan-Envigo TD.88137, 21% fat and 0.2% cholesterol by weight) for 16 weeks starting at 12 weeks old of age for 16 weeks starting at 12 weeks old. Body weight was measured and 4 h fasting blood were collected every month. Partial carotid ligation (PCL) model PCL surgery was performed as published. 41 , 65 The left external carotid artery (CA), left internal CA, and left occipital artery were ligated, while the left superior thyroid artery was not. Ligated and non-ligated arteries were collected 1 week after the surgery. EC isolation Liver EC isolation was performed as described with modifications. 66 Briefly, mice were humanely euthanized, the vascular system washed with PBS via cardiac perfusion and livers were collected. In a small tube, the liver tissue was chopped into small pieces and blood cells were lysed using ACK lysis buffer. Next, tissue was digested in collagenase type I at 37°C for 60 minutes. ECs were enriched by using mouse anti-CD146 and CD144 antibodies (REAfinity, Miltenyi Biotec, CA, USA). For aortic ECs, aortas were collected after perfused with PBS and then flushing with TRIzol reagent (Thermo Fisher Scientific), as described 65 . Plasma biochemical index testing Total, HDL, and LDL/VLDL-cholesterol were measured in serum using the HDL and LDL/VLDL Quantitation Kit (Sigma-Aldrich #MAK045; #CS0005) following manufacturer’s instructions. Specifically, 15-50 µl of serum was fractionated by adding equal volume of fractionating reagent (2x LDL/VLDL Precipitation Buffer Sigma#MAK045B) and then centrifugation at 12,500g for 10 minutes to yield the HDL supernatant phase and a pellet containing the low-density lipoproteins (LDL and VLDL). These fractions were used to measure cholesterol, and the values added to obtain the total circulating cholesterol. ALT was measured in serum using the Liquid ALT kit (Pointe Scientific # A7526-01-1953), and AST was measured in serum using the Aspartate Aminotransferase Colorimetric Activity Assay kit (Cayman #701640). The change in absorbance in both assays was recorded for 40 minutes at 37°C, calculating the activity from the slope from a minimum linear period of 10 minutes. Atherosclerotic and liver lesion analysis After humane euthanasia, mice were perfused with 20 mL 0.01 M PBS through the left ventricle. The total aorta and heart were harvested and fixed. The en face aortic root preparation and quantification of the lesion areas in the whole aorta were performed as described. 67 For en face analysis, the whole aorta was cut open and stained with oil red O (ORO) (Sigma). Lesion in the aortic arch (aortic root to below the left subclavian artery), thoracic aorta (the region between the end of the arch and the last intercostal branch) and abdominal aorta (the region between the end of the thoracic aorta segment and the iliac bifurcation) were assessed. For aortic root analysis, the heart was embedded in OCT, snap-frozen in liquid nitrogen, and cross-sectioned serially at the aortic root level at a 7 μm thickness. The cryosections were stained with ORO and counterstained with hematoxylin. Images were taken using Zeiss Observer II microscope, and quantification was determined with ImageJ. Livers from mice were collected, embedded in OCT and sectioned with a cryostat. Slides were fixed in 4% (vol/vol) paraformaldehyde (PFA) for 10 minutes for histological analyses. Histology and immunohistochemistry staining Hematoxylin & Eosin staining (H&E) Tissues were harvested and fixed in 10% neutral buffered formalin. Dehydration, clearing, and paraffinization was performed on a Tissue-Tek VIP Vacuum Infiltration Processor (SAKURA). The samples were embedded in paraffin using a Tissue-Tek TEC Tissue Embedding Station (SAKURA) and sectioned and put on positively charged glass slides. The slides were deparaffinized, rehydrated, and stained with Modified Mayer’s Hematoxylin and Eosin Y Stain (America MasterTech Scientific) on a H&E Auto Stainer (Prisma Plus Auto Stainer, SAKURA) according to standard laboratory procedures. Immunohistochemistry (IHC) The IHC stains were performed using a Ventana Discovery Ultra IHC automated stainer (Ventana Medical Systems, Roche Diagnostics, Indianapolis, USA). Slides were incubated with endogenous peroxidase activity inhibitor and antigen retrieval solution. Anti-mouse F4/80 (Cat#: 70076, Cell Signaling, 1:2000), or alpha-smooth muscle actin (Cat#: ab124964, Abcam, 1:4000) were incubated followed by DISCOVERY anti-rabbit HQ and DISCOVERY anti-HQ-HRP (Ventana). The antibodies were visualized by DISCOVERY ChromoMap DAB Kit (Ventana) and counterstained with hematoxylin and cover slipped. Whole slide images were acquired with a NanoZoomer S360 Digital Slide Scanner (Hamamatsu) and viewed by NDP.view image viewer software. Masson’s trichrome staining The stains were performed on a BenchMark Special Stains Instrument (Ventana Medical Systems, Roche Diagnostics, Indianapolis, USA) using a Trichrome staining kit (06521908001). The slides were then rinsed, dehydrated, and mounted. Stimulated Raman Scattering (SRS) microscopy configuration, ratiometric image processing, and data analysis The instrument setup of the SRS microscope was described. 68 Briefly, SRS images were acquired using an 80 μs pixel dwell time that resulted in an acquisition speed of 8.52 s/frame for a 320x320 pixel field of view. The pump beam wavelength was set to 791.3 nm for the –CH 3 channel (2940 cm-1), 797.3 nm for the –CH 2 channel (2845 cm⁻1), and 938.3 nm for the phosphate channel (960 cm-1). Laser powers for the liver samples were set to 100 mW for the Pump beam and 100 mW for the modulated Stokes beam. For the aorta samples, the Stokes and Pump powers were set to 100 mW and 100 mW for the protein channel, and 100 mW and 300 mW for the lipid and phosphate channels. Image analysis and color coding were performed using ImageJ. Linear unmixing of the protein (-CH 3 ) and lipid (-CH 2 ) channels was performed in MATLAB (R2024b, MathWorks) using the method as reported. 69 For ratiometric analysis, a mask image was generated by adjusting the threshold and normalizing non-zero values to one using the CH 3 image. Unmixed CH 2 images were then divided by corresponding unmixed CH 3 images from the same set, and the resulting ratiometric image was multiplied by the mask image to produce the final CH 2 :CH 3 ratiometric image. ASO design and synthesis and LNP-ASO formulation The ASOs were designed based on the homologous regions of human and mouse AGO1 mRNA transcripts. ASOs are 16 nt-long gapmers chemically modified for nuclease resistance using fully phosphorothioated backbone and locked nucleic acid (LNA) modification of 1-3 nt at both 5’ and 3’ ends. 70 Sequences are provided in Table S1. ASO gapmers were synthesized and at the DNA/RNA Synthesis Laboratory (City of Hope) and were purified and analyzed using anion-exchange HPLC, desalted and lyophilized. A scrambled control LNA Gapmer was purchased from QIAGEN with a sequence of 5’-TAACACGTCTATACGC-3’ (Product #339516). The lipid nanoparticles were synthesized using a microfluidic device (NanoAssemblr Ignite, Precision NanoSystems). All lipids, including SM102 (Echelon Bioscience, Cat#N1102) Cholesterol (Avanti Polar Lipids, Cat#700000), DSPC (Avanti Polar Lipids, Cat#850365), and DMG-PEG (Avanti Polar Lipids, Cat#880151), were dissolved in ethanol and mixed at a mole ratio of 50:38.5:10:1.5, respectively. The AGO1-ASO was dissolved in citrate buffer (pH-4, 50 mM). The aqueous and ethanol phases were mixed at a volumetric ratio of 3:1 using a microfluidic device. The formulated LNP-ASO were then dialyzed in 1×PBS using a dialysis bag with a molecular weight cutoff set at of 12-14kDa (Spectra/Por, Part# 132703T) for 2 hours, then analyzed for encapsulation efficiency, size (97.64 nm), and zeta potential (-0.5 mV). Cell culture, treatment, and transfection Cells were kept at 37°C ventilated with 5% CO 2 and 21% oxygen. HAECs (at passages 5–8) or HLSECs (within passage 3) or HUVECs (passages 5–8) were cultured in complete M199 medium supplemented with 20% FBS (Sigma, M2520) and 1× antibiotics (penicillin– streptomycin, Gibco, 15140122). HepG2 cells were cultured in DMEM with 10% FBS. In some experiments, cells were treated with oxPAPC (40 μg/mL) (Avanti Polar Lipids, Cat# 870604) or vehicle control for 4 hours. In some experiments, cells were pre-transfected with ASOs using Lipofectamine RNAiMax as described. The medium was changed to M199 containing 15% FBS 4-6 hours after transfection. Luminex assay Cultured medium (500 µl in triplicates) was collected and subjected to Luminex assay using a curated human 14-plex panel (NB1097; Bio-techne) at Analytical Pharmacology Core at City of Hope. Monocyte adhesion assay Monocytes were labeled with CellTracker Green CMFDA Dye (Thermo Fisher Scientific) and incubated with a monolayer of HAEC or HUVECs (4 × 10 3 cells per cm 2 ) for 20 minutes. The non-attached monocytes were then washed off with complete EC growth medium. The attached monocyte numbers were imaged using an Echo Revolve microscope under the 10x lens and green fluorescent channel. Average numbers per condition were calculated using a counter on ImageJ in a blinded fashion from five randomly selected fields of technical duplicates. Cell fractionation Cell fractionation was performed as described. 71 Briefly, 5-10 million cells were collected, washed with 1 mL of cold PBS containing 1 mM EDTA, and centrifuged at room temperature at 500 × g and the cell pellet collected. A small portion (10%) of the cell pellet was set aside as the input (total fraction). The remaining pellet was suspended in 200 μL of ice-cold lysis buffer (10 mM Tris-HCl, pH 7.5, 0.05% NP-40, 150 mM NaCl) and incubated on ice for 5 minutes. The lysate was gently pipetted over 2.5 volumes of chilled sucrose cushion (24% RNase-free sucrose in lysis buffer) and centrifuged at 15,000 × g for 10 minutes at 4 °C. The supernatant, representing the cytoplasmic fraction, was collected. The pellet was suspended in 50 μL of the RIPA buffer to obtain the nuclear fraction. Protein concentrations were quantified using the BCA method, and SDS-PAGE was subsequently performed. Cytoplasmic and nuclear markers, β-tubulin (CST, cat # 2416) and Lamin B1 (CST, cat # D9V6H), were used for validation. RNA extraction, quantitative PCR, and RNA-seq Total RNA was extracted from tissues and cells using TRIzol reagent (Invitrogen, cat # 15596026). Extraction from tissue had an additional first step of bead homogenization. cDNAs were synthesized using PrimeScript™ RT Master Mix containing both Oligo-dT primer and random hexamer primers. qPCR was performed with Bio-Rad SYBR Green Supermix using the Bio-Rad CFX Connect Real Time system. 36B4 was used as internal control in mouse and β-actin or 18S in human samples. The primers used in this study are summarized in Table S3. For RNA-seq, 1 µg of total RNA from three biological replicates was used for library preparation with the KAPA mRNA HyperPrep Kit (Roche, cat # KK8581). mRNA was enriched using mRNA capture beads, and libraries were prepared following the manufacturer’s protocol (KR0960-v6.17). The quality of cDNA libraries was assessed using the Agilent 4200 TapeStation System. Libraries were sequenced using the NovaSeq platform (Illumina) with 150-nt paired-end sequencing, achieving a depth of 50∼100 million read pairs. Cut&Tag-seq and Chromatin immunoprecipitation (ChIP) Cells were collected, centrifuged at 600 × g for 5 minutes, and the cell pellets retained. For each sample, 500,000 cells were used to prepare libraries following the EpiCypher Cut&Tag protocol with CUTANA™ Cut&Tag Kit (EpiCypher, cat # SKU: 14-1102). Briefly, 10 µL of ConA beads per sample were activated using bead activation buffer. The activated beads were incubated with the cell pellets at room temperature for 10 minutes to bind the cells. Subsequently, 0.5 µg of primary antibody was added to each sample and incubated at 4 °C overnight. IgG (EpiCypher, cat # SKU:13-0024) (negative control) and H3K27ac (EpiCypher, cat # SKU: 13-0059) (positive control) antibodies were used. On the second day, pAG-Tn5 was added to each reaction, and tagmentation buffer was used to activate the Tn5 enzyme for chromatin fragmentation. Following tagmentation, CUTANA high-fidelity 2× PCR Master Mix was used to amplify the DNA fragments under the recommended parameters. Targeted chromatin tagmentation was completed following the epicypher protocol. Libraries were amplified with 14 PCR cycles and purified by single sided 1.3x AMPure bead purification Finally, cDNA quality was assessed using an Agilent High Sensitivity DNA chip. Libraries were sequenced on the NovaSeq platform (Illumina) with 150-nt paired-end sequencing, achieving a depth of 5–10 million read pairs. For ChIP, cells were crosslinked with 1% formaldehyde (Sigma Aldrich, F8775) for 15 min at room temperature, quenched with 0.16 M glycine (pH 7.0) for 5 min at 4°C, and washed in cold PBS. Fixed cells were sequentially washed at 4°C for 10 min with ChIP buffer A (10 mM HEPES, pH 7.6, 10 mM EDTA, pH 8.0, 0.5 mM EGTA, pH 8.0, and 0.25% Triton X-100), followed by ChIP buffer B (10 mM HEPES, pH 7.6, 100 mM NaCl, 1 mM EDTA, pH 8.0, 0.5 mM EGTA, pH 8.0, and 0.01% Triton X-100). Cell pellets were resuspended in sonication buffer (15 mM HEPES, pH 7.6, 140 mM NaCl, 1 mM EDTA, pH 8.0, 0.5 mM EGTA, pH 8.0, 0.1% sodium deoxycholate, and 1% Triton X-100) supplemented with Complete Mini EDTA-free protease inhibitors (Roche), 0.5% SDS, and 0.2% N-lauroylsarcosine, at a final concentration of 1∼5 × 10⁷ cells/mL. Chromatin was sheared using a Bioruptor (Diagenode) at high power to generate DNA fragments of 200∼500 bp. The sonicated chromatin was centrifuged at 12,000 rpm for 10 min at 4°C and diluted fivefold to reduce detergent concentrations. Immunoprecipitations were carried out by incubating the sonicated chromatin with a p65 antibody (CST, cat # 6956S, 1∶500) or a rabbit IgG overnight at 4°C. Equal amounts of Protein A and G Dynabeads (Invitrogen), pre-blocked with BSA (1 mg/mL), were added to the antibody-chromatin mixture and incubated for 4h at 4°C. The beads were then subjected to sequential 10-min washes with Wash A (10 mM Tris, pH 8.0, 1 mM EDTA, pH 8.0, 140 mM NaCl, 0.1% SDS, 0.1% sodium deoxycholate, and 1% Triton X-100), Wash B (Wash A adjusted to 500 mM NaCl), Wash C (10 mM Tris, pH 8.0, 1 mM EDTA, pH 8.0, 250 mM LiCl, 0.5% sodium deoxycholate, and 0.5% IGEPAL CA-630), and TE buffer. Beads were resuspended in 100 μL TE, treated with RNase A (20 μg/mL) at 55°C for 30 min, supplemented with 0.75% SDS and 50 mM Tris, and subjected to overnight reverse crosslinking at 65°C. The eluted DNA was then treated with Proteinase K at 55°C for 2 h and purified using phenol-chloroform extraction. Enrichment fold was determined by using the 2 −ΔCq method relative to input controls. Seq data analysis RNA-seq data were analyzed as we previously described. 26 Raw RNA-seq reads were trimmed to remove adaptors using Trimmomatic (v.0.39) with default settings. Processed reads were aligned to the human reference genome (hg38) using STAR (v.2.7.10) with default parameters. Gene expression was quantified as transcripts per million (TPM) using featureCounts from the Subread package (v.2.0.1). Genes with expression levels below 10 reads were excluded. Fold changes were calculated as the ratio of expression levels in treatment versus control samples. Differential expression analysis was conducted using DESeq2. Genes with false discovery rate (FDR) = 0.5 and downregulated for fold changes <=-0.5. The Human MSigDB v2024.1.Hs dataset was downloaded from the GSEA database, and genes were ranked by log fold change in descending order. GSEA enrichment analysis was performed using the clusterProfiler R package. Reads were converted to TPM and scaled using z-scores for heatmap plotting. Cut&Tag data analysis was performed as published. 72 Raw sequencing reads with low quality and adaptor sequences were trimmed using Trimmomatic (v.0.39) with default parameters. Trimmed reads were aligned to the hg38 reference genome using Bowtie2 with the parameters “--local --very-sensitive-local --no-mixed --no-discordant”. Duplicate reads were removed using Picard, and the aligned reads were sorted using Samtools. Normalized bigWig files were generated from BAM files using Deeptools with the “–normalizeUsing RPKM” parameter. For peak calling, MACS2 was used with the following parameters: “callpeak --keep-dup all --p 1e-5 --f BAMPE”. Peaks overlapping blacklist regions were removed using Bedtools intersect. Filtered peaks were annotated to the nearest genes from the peak center using the annotatePeaks.pl script from HOMER. Promoter regions were defined as regions within 3 kb of the transcription start site (TSS). Prior to creating bigWig track files, BAM files were converted to BED format using Bedtools bamtobed. Genome-wide visualization of bigWig files and called peaks was performed using the WashU Genome Browser. Heatmap analysis was conducted using the Deeptools package. Transcription factor enrichment and motif analyses were performed with the HOMER v5.0 software package. Western blotting, co-IP, and (co-)IF Western blotting Tissues or cells were homogenized in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1 mM DTT, 1% NP-40, 0.1% SDS, and 1× protease inhibitor cocktail). Proteins were separated by SDS-PAGE and transferred onto PVDF membranes. Membranes were processed according to the ECL western blotting protocol (ThermoFisher). Images were captured using the Amersham Imager 680 (GE Healthcare). The gray values of Western blot bands were quantified using ImageJ. Antibodies included mouse AGO1 monoclonal antibody (2A7) (WAKO, cat # 015-22411), rabbit AGO1 polyclonal antibody (ThermoFisher, cat# PA5-50654), rabbit β-tubulin (CST, cat # 2144), rabbit NF-κB (D14E12) (CST, cat # 8242), rabbit p-AMPKα (Thr172) (40H9) (CST, cat # 2535), rabbit phospho-acetyl-coA carboxylase (Ser79) antibody (CST, cat # 3661), lamin B1 (D9V6H) Rabbit mAb (CST, cat # 13435). Co-IP For co-IP, cells were lysed in RIPA buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, and 0.1% NP-40) supplemented with protease and phosphatase inhibitors. The lysates were incubated overnight at 4°C with Protein A/G agarose beads and AGO1 antibody (Thermo Fisher, cat # PA5-50654, 1:1000) or p65 antibody (CST, cat # 8242S, 1:500), while rabbit IgG antibody served as a control. After extensive washing with lysis buffer, the immunoprecipitates were eluted and subjected to SDS-PAGE, followed by Western blot analysis. (Co-)IF For (co-)IF, ECs seeded on coverslips were washed with PBS and fixed in 4% paraformaldehyde at room temperature for 15 minutes. Cells were permeabilized in PBS containing 0.3% Triton-X-100 for 10 minutes, rinsed with PBS, and blocked with 10% goat serum at room temperature for 1 hour. Coverslips were incubated with primary antibodies anti-AGO1 (Thermo Fisher, cat # PA5-50654, 1∶1000) only or also with anti-p65 (CST, cat # 6956S, 1∶1000) diluted in 10% goat serum overnight at 4°C. Cells were washed with PBS and subsequently treated with anti-Rabbit Alexa Fluor™ 594 (Thermo Fisher, cat # A-11012, 1∶1000) or anti-mouse Alexa Fluor™ 488 (Thermo Fisher, cat # A-10680, 1∶1000) secondary antibodies at room temperature for 1 hour. Coverslips were washed and mounted with mounting media containing DAPI. IF images were captured using a Zeiss confocal microscope and a PerkinElmer Operetta CLS High Content Analysis imaging system. Ten to 20 cells from 2-3 randomly selected fields were quantified using Fiji by two independent researchers in a blinded manner. Statistics All in vitro data represent at least three independent experiments unless otherwise indicated. All in vivo data represent experiments performed with numbers of mice as specified in the figure legends. Statistical analyses for data other than high-throughput sequencing (see Methods in the Data Supplement) were performed using GraphPad Prism. Two-group comparisons were performed using 2-sided Student’s t-test and multiple-group comparisons were performed using ANOVA followed by Tukey’s post-hoc test. P values less than 0.05 were considered statistically significant. Data availability All newly generated sequencing data are available at GEO numbers for Cut&Tag: GSE293927 and bulk RNA-seq: GSE293932. Sources of Funding This work was supported by NIH grants R01HL145170, R35HL171550 (to Z.B.C.), R01CA284593 (to M.K.), R01AI153064 (to N.P.) and American Heart Association Postdoctoral fellowships 24POST1195441 (to X.L.), 25POST1365287 (to N.K.M.) and California Institute of Regenerative Medicine grant EDU4-12772 (to A.T.). L.W. is a Heritage Principal Investigator supported by the Heritage Medical Research Institute at California Institute of Technology, and further acknowledges the support received from a CZI Dynamic Imaging grant. Research reported in this publication included work performed in the Comprehensive Mouse Phenotyping, Integrative Genomics, DNA/RNA Synthesis, and Light Microscopy and Digital Imaging, and Pathology Cores supported by the National Cancer Institute of the NIH under award number P30CA033572. Disclosures None. Download figure Open in new tab Figure S1. EC-AGO1-KO improves plasma lipid profiles in a diet-induced obesity model. Starting at 8 weeks old, male EC-AGO1-KO mice and WT littermates were fed a HFHS diet for 16 weeks (n=4/group). A-D, Plasma triglyceride, total cholesterol, LDL/VLDL, and HDL levels after 4 hours fasting. Data are presented as mean±SEM. *p<0.05 based on Student’s t-test. Download figure Open in new tab Figure S2. Comparable levels of AGO1 expression in monocytes and AGO2 expression in ECs from the EC-AGO1-KO vs WT littermates. qPCR analysis of mRNA levels of AGO1 in bone marrow-derived monocytes (in A ) and AGO2 in ECs isolated from the liver and aorta (in B ) from 12-week-old male EC-AGO1-KO and their WT littermates (n=3-4/group). 36B4 was used as an internal control. Data are presented as mean±SEM. Download figure Open in new tab Figure S3. EC-AGO1-KO show no effect on AAV transduction efficiency. Male EC-AGO1-KO mice and WT littermates were injected with the same dose of AAV9-tdTomato through tail-vein and the livers were collected 1-week later. mRNA levels of Tdtomato were quantified by qPCR with 36B4 as the internal control (n=6/group). Data are presented as mean±SEM. Download figure Open in new tab Figure S4. Dose-dependent expression of AAV9-PCSK9. WT mice were injected with PBS or 1×, 3×, and 5×10 11 viral genome (vg) of AAV9-PCSK9. Livers were collected 1-week later. mRNA levels of PCSK9 were quantified by qPCR with 36B4 as the internal control. Download figure Open in new tab Figure S5. Body weight of EC-AGO1-KO and WT littermates in the AAV-PCSK9 induced AS mice model. Body weight of mice described as in Figure 1 (n=5-7/group). Data are presented as mean±SEM. Download figure Open in new tab Figure S6. EC-AGO1-KO maintains metabolic homeostasis in AAV-PCSK9-exposed mice. Male EC-AGO1-KO mice and WT littermates were injected with one dose of AAV-PCSK9 and fed under chow diet for 16 weeks starting at 12 weeks old (n=9 WT and 8 KO). A , Body weight measured monthly from the AAV-PCSK9 administration. B , Representative picture of plasma samples from two groups of mice after 4 hours fasting. C-F , Plasma triglyceride, total cholesterol, LDL/VLDL and HDL after 4 hours fasting. G , Relative liver weight. H-I , Plasma ALT and AST levels after 4 hours fasting. Data are presented as mean±SEM. *p<0.05, **p<0.001 based on Student’s t-test. Download figure Open in new tab Figure S7. EC-AGO1-KO attenuates atherosclerosis in different segments of aorta in the AAV-PCSK9 and atherogenic diet exposed mice. Mice were the same as in Figure 1 . Quantification of plaque areas in different segments of aorta. Data are presented as mean±SEM. **p<0.01, ***p<0.001 between the indicated groups based on one-way ANOVA. Download figure Open in new tab Figure S8. Knockdown (KD) effect of AGO1-ASO in ECs. HUVECs were transfected with a scrambled (Scr) or one of the three AGO1 ASOs (i.e., 110, 111 and 112) at 20, 50 and 100 nM (left to right). AGO1 mRNA levels were quantified by qPCR, with b-actin as the internal control. Data represents mean±SEM. Download figure Open in new tab Figure S9. AGO1-KD attenuates monocyte adhesion to HUVECs. HUVECs were transfected with Scr or AGO1-ASO (20 nmol/L) and then treated (HT) for 48 hours for monocyte adhesion assay. Representative images (in A ) and quantification (in B ) of monocytes attachment to HUVECs. Scale bar = 200 µm. Data are presented as mean±SEM. *p<0.05, **p<0.01 based on two-way ANOVA. Download figure Open in new tab Figure S10. Effect of AGO1 inhibition on the hepatocytes. HepG2 cells with or without AGO1-KD were treated with TNF-α (5 ng/mL) for 24 h. qPCR analysis of mRNA levels of marker genes associated with inflammation and lipid metabolism. Data are presented as mean±SEM. **p<0.01 based on Student’s t-test. Download figure Open in new tab Figure S11. Effect of EC-AGO1-KO in livers from mice given a chow diet. Male EC-AGO1-KO mice and their WT littermates were fed a chow diet for 28 weeks (n=7/group). qPCR of liver mRNA and analysis of levels of genes involved in inflammation and lipid metabolism in the liver of mice. Data are presented as mean±SEM. *p<0.05, **p<0.001 based on Student’s t-test. Download figure Open in new tab Figure S12. Co-IF of AGO1 and NF-κB in HUVECs. A , Representative confocal microscopy images of AGO1 (red) and p65 (green) in HAECs treated with HT. The arrow in each merged image indicates the plane for generating line profiles and calculation of Pearson’s correlation coefficient (r) ( B, C ). D , The co-localization of AGO1 and p65 in the nucleus were quantified in 10 cells per group. Scale bar = 20 mm. **p<0.01 based on Student’s t-test. Download figure Open in new tab Figure S13. ASO and LNP-ASO AGO1 knockdown efficiency test in mice. A , WT mice were injected i.v. with PBS, Scr, or AGO1-ASO at the dose of 10, 20, or 30 mg/kg body weight. A week later, livers were harvested and mRNA levels of Ago1 were quantified by qPCR in livers after one week. B , WT mice were injected i.v. with PBS, Scr, AGO1-ASO (10 mg/kg body weight), or LNP-AGO1-ASO (1 mg/kg dose). A week later, livers were harvested and mRNA levels of Ago1 were quantified by qPCR. n=2-3/group. Data are presented as mean±SEM. Download figure Open in new tab Figure S14. LNP delivery of AGO1 does not show liver toxicity in mice. Mice were treated as in Fig. 6 (n=5 per group). A , Relative liver weight. B, C , Plasma ALT and AST levels. Data are presented as mean±SEM. Download figure Open in new tab Figure S15. Effect of EC-AGO1-KO and AGO1-ASO in female mice treated by atherogenic regimen. A-E , Female mice were under the same treatment as in Figure 1 , i.e., injected with AAV9-CTRL/PCSK9 and fed a high fat high cholesterol diet (HFHCD) for 16 weeks (n=5 per group). A , Body weight. B-E , Levels of TG, TC, LDL/VLDL and % HDL/total cholesterol in the plasma after 4 hours fasting. F-H , Mice were under the same treatment as in Fig. 6 , i.e., received AAV9-PCSK9 and fed a HFHCD. Two weeks later, LNP-ASO was injected through tail vein at 1 mg/kg body weight weekly for 4 consecutive weeks (n=5 per group). F , Body weight. G, H , mRNA levels of Icam1 and Ccl2 were quantified by qPCR in livers ( G ) and aortas ( H ). Data are presented as mean±SEM. *p<0.05, **p<0.01 based on Students t-test. View this table: View inline View popup Download powerpoint Table S1. Sequences of AGO1 ASOs View this table: View inline View popup Download powerpoint Table S2. AGO1-regulated genes that show AGO1-binding in the promoters View this table: View inline View popup Table S3. Sequences of primers used for PCR Acknowledgments The authors wish to thank Dr. Yun Fang at University of Chicago, for the generous gift of AAV9-PCSK9 and Drs. Zhao Wang, Holly Yin, and Jiawei Sun and Ms. Muxi Chen at the City of Hope, Dr. Samar Ibrahim at Mayo Clinic, and Drs. Shu Chien and John Shyy at the University of California San Diego for the valuable discussion, and Dr. Jefferey Isenberg at City of Hope for the helpful editing. Z.B.C. conceived the study, acquired funding, and supervised the experiments and analyses. X.L. and Z.B.C. designed the experiments and wrote the manuscript. X.L. performed the majority of mouse and cell experiments and the related data analyses. Y.L., P.S., and M.K. designed the AGO1-ASO. Y.L. and X.T. performed the initial in vitro and in vivo tests of the ASO. X.T. performed the lipid profiling in HFHS diet-fed animals. Y.L. prepared the bulk RNA-seq library. 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Share Endothelial AGO1 Drives Vascular Inflammation and Atherosclerosis via a Non-Canonical Nuclear Mechanism Xuejing Liu , Dongqiang Yuan , Yingjun Luo , Xiaofang Tang , Alonso Tapia , Naseeb Kaur Malhi , Rahuljeet Singh Chadha , Sachchidanand Tiwari , Piotr Swiderski , Kuei-Chun Wang , Marcin Kortylewski , Norbert Pardi , Lu Wei , Wendong Huang , Zhen Bouman Chen bioRxiv 2025.05.01.651783; doi: https://doi.org/10.1101/2025.05.01.651783 Share This Article: Copy Citation Tools Endothelial AGO1 Drives Vascular Inflammation and Atherosclerosis via a Non-Canonical Nuclear Mechanism Xuejing Liu , Dongqiang Yuan , Yingjun Luo , Xiaofang Tang , Alonso Tapia , Naseeb Kaur Malhi , Rahuljeet Singh Chadha , Sachchidanand Tiwari , Piotr Swiderski , Kuei-Chun Wang , Marcin Kortylewski , Norbert Pardi , Lu Wei , Wendong Huang , Zhen Bouman Chen bioRxiv 2025.05.01.651783; doi: https://doi.org/10.1101/2025.05.01.651783 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Pathology Subject Areas All Articles Animal Behavior and Cognition (7618) Biochemistry (17636) Bioengineering (13860) Bioinformatics (41847) Biophysics (21401) Cancer Biology (18536) Cell Biology (25424) Clinical Trials (138) Developmental Biology (13353) Ecology (19860) Epidemiology (2067) Evolutionary Biology (24287) Genetics (15583) Genomics (22463) Immunology (17701) Microbiology (40300) Molecular Biology (17141) Neuroscience (88434) Paleontology (666) Pathology (2825) Pharmacology and Toxicology (4813) Physiology (7633) Plant Biology (15107) Scientific Communication and Education (2042) Synthetic Biology (4285) Systems Biology (9808) Zoology (2268)