Introduction: Ionizing radiation (IR) promotes cellular senescence, DNA damage, impaired efferocytosis, and dysregulation of clonal hematopoiesis (CH) drivers, collectively accelerating radiation-induced atherosclerosis (AthS); however, the mechanisms by which colchicine modulates these processes remain unclear. Hypothesis: Colchicine attenuates IR-induced inflammation and senescence, including CH-associated pathways, thereby limiting AthS progression. Methods: Bone marrow-derived macrophages (BMDMs) were pretreated with low-dose colchicine and exposed to 2 Gy IR. RNA sequencing and Western blot analyses assessed molecular changes. IR-induced senescence was assessed using a CellAge-based composite senescence score together with GSEA and GO pathway enrichment analyses. Structual docking was performed using AutoDock Vina v1.2.5 employing the default vina scoring function. In vivo effects of colchicine on radiation-induced AthS were evaluated using a partial carotid ligation model, with spatial proteomic changes assessed by CO-Detection by Indexing (COMET) analysis. Results: Colchicine did not broadly suppress IR-induced inflammatory gene expression but robustly restored Aldehyde Dehydrogenase (ALDH)1/2, which was markedly downregulated by IR. While NF-κB activation was only partially reduced, colchicine significantly decreased inflammatory cytokine secretion and suppressed senescence phenotypes, including SA-β-gal activity, mitochondrial reactive oxygen specoes, Nuclear Factor Erythroid 2–Related Factor loss, and NAD/ATP depletion, accompanied by reduced senescence transcriptional signatures. Colchicine did not inhibit early IR-induced p90RSK activation, indicating a downstream mechanism. Pharmacologic ALDH2 activation (Alda-1) recapitulated, whereas ALDH2 inhibition (CVT-10216) abolished, colchicine’s anti-senescent effects. Docking and in tube enzyme assays demonstrated direct, dose-dependent activation of ALDH2 by colchicine. In vivo, colchicine attenuated IR-accelerated AthS and senescence markers. After radiation therapy (RT), human monocyte-derived macrophages showed reduced ALDH2 and accumulation of downstream 4-Hydroxynonenal (4-HNE) , indicating impaired aldehyde detoxification and oxidative stress in cancer patients after RT. Conclusion: Colchicine directly activates ALDH2 to suppress radiation-induced senescence and inflammation, revealing a novel mechanism and therapeutic strategy for radiation-associated cardiovascular disease in cancer patients.

Introduction: Atherosclerotic lesions preferentially form in arterial regions exposed to disturbed flow (DF), where ECs exhibit mitochondrial (mt) dysfunction, metabolic reprogramming, and senescence. Hippo pathway kinases LATS1/2 maintain EC homeostasis, but how DF–mediated LATS1/2 suppression drives EC metabolic remodeling remains unclear. Hypothesis: We propose that DF activates CD38, leading to mt dysfunction and metabolic reprogramming that promotes EC senescence and aberrant proliferation by LATS1/2 inhibition. Methods: EC-specific Lats1/2 knockout mice were generated using tamoxifen-inducible Cre systems, and DF was induced by partial carotid ligation. Murine and human plaques were analyzed using COMET™ immunofluorescence, and spatial metabolomics. Bioenergetic profiling included Seahorse XF analysis, 13 C-glucose/ 13 C-glutamine tracing, and pharmacologic modulation of CD38, mtROS, and ATP synthase. Results: Complete Lats1/2 deletion caused fatal edema and vascular leakage, while partial loss produced fragile, highly neovascularized plaques. Spatial proteomics revealed a CD38-driven senescence-associated stemness (SAS) phenotype under LATS1/2 deficiency. Metabolomics showed sulfite and taurine accumulation, consistent with SUOX deficiency. Mechanistically, CD38 suppressed SUOX, induced Complex V reverse-mode , increased succinate flux, and accelerated ATP consumption. Despite ATP depletion, glutamate metabolism and TCA cycle flux were enhanced, sustaining EC proliferation under energetic stress. LATS1/2 knockdown reduced mt respiratory capacity (lower OCR peak) without significantly increasing glycolysis, and DF suppressed both OCR and ECAR, indicating impaired energy metabolism under pathological shear stress. CD38 inhibition restored mt respiration and glycolytic capacity by preserving NAD, and rescuing LATS1/2 and DF-mediated defects. Blocking reverse-mode Complex V reduced DF- and LATS1/2-dependent EC senescence and proliferation. Conclusion: Complete LATS1/2 deletion causes fatal vascular leakage, while partial loss drives plaque formation with fragile neovessels through a CD38-dependent SAS phenotype. CD38 suppresses SUOX, induces Complex V reverse-mode , increases succinate flux, and accelerates ATP consumption, enabling EC proliferation despite energy stress. DF and LATS1/2 loss impair mt and glycolytic function, whereas CD38 inhibition restores mt respiration and reduces EC senescence and proliferation to prevent atherothrombosis in DF regions.

Background: Ionizing radiation (IR) is a well-recognized contributor to cardiovascular disease, in part through endothelial cell (EC) injury characterized by oxidative stress, DNA damage, telomere dysfunction, and premature cellular senescence. These processes drive chronic inflammation and accelerate atherosclerosis (AthS). Although the serine/threonine kinase p90RSK regulates multiple stress-responsive and inflammatory pathways, its role in IR-induced chromosomal instability and long-term vascular injury remains incompletely understood. Hypothesis: Pharmacologic Inhibition of p90RSK following irradiation mitigates IR-induced DNA damage and ECs senescence by restoring chromosomal and telomere stability, thereby attenuating IR-accelerated AthS. Methods: Human Umbilical Vein Endothelial Cells were exposed to 5 Gy X-radiation and treated with p90RSK inhibitors (FMK-MEA or BI-D1870). Genomic instability and DNA damage were assessed using dicentric chromosome assays, micronucleus analysis, chromosome orientation–fluorescence in situ hybridization, and neutral comet assays. Transcriptomic changes were evaluated by RNA sequencing. In vivo relevance was examined using a partial carotid ligation model of radiation-accelerated AthS. Results: IR induced marked increases in chromosomal aberrations, telomere instability, and DNA double-strand breaks in ECs, accompanied by sustained activation of senescence-associated and pro-inflammatory transcriptional programs. Post-IR inhibition of p90RSK significantly reduced both acute and persistent genomic damage, restored telomere stability, and suppressed EC senescence and senescence associated secretory phenotype. In vivo, p90RSK inhibition attenuated IR-mediated acceleration of AthS. Transcriptomic analyses identified KCNK6 , a potassium channel linked to inflammatory signaling, as a novel and downstream effector uniquely reversed by p90RSK inhibition. Genetic depletion of KCNK6 recapitulated the protective effects of p90RSK inhibition, reducing IR-induced senescence and inflammatory signaling. Conclusion: p90RSK serves as a central mediator of IR-induced endothelial genomic instability, linking DNA damage to cellular senescence and AthS progression. Pharmacologic inhibition of p90RSK provides durable vascular radioprotection by preserving chromosomal and telomere integrity and suppressing inflammatory remodeling, identifying p90RSK and KCNK6 as therapeutic targets for radiation-associated cardiovascular disease.

AIMS:Radiation therapy (RT) is an integral component of cancer therapy but associated with adverse events. Our goal was to establish risk prediction models for major adverse cardiovascular and cerebrovascular events (MACCE) after chest RT. METHODS AND RESULTS:A retrospective study of lung/breast cancer patients who had chest RT with planning CT at Mayo Clinic between 01/2010 and 01/2014. Predictive models were developed based on weighted independent predictors using a derivation (406 lung and 711 breast cancer) and validation cohort (179 lung and 234 breast cancer). Patient characteristics, pre-RT CT for coronary artery calcification (CAC), and post-RT MACCE data were reviewed. Post-RT MACCE occurred in 6.1 and 5.6% in the derivation and validation cohort over a mean follow-up of 42 ± 13 months. Post-therapy model (C2AD2) included CAC (two points), MACCE history (two points), age ≥74 (three points), DM (two points), and mean heart radiation dose ≥ 850 mGy (two points), and pre-therapy model (C2AD) included post-therapy model parameters minus mean heart radiation dose. Both models stratified patients into three risk groups: low (0-2), intermediate (3-5), and high (≥6). Post-RT MACCE across these groups were 2.7, 8.9, and 19.8% in the derivation, and 3.9, 6.6, and 16.4% in the validation cohort for post-therapy model (C2AD2) and 2.8, 9.2, and 20.4% in the derivation and 3.7, 9.2, and 13.2% in the validation cohort for pre-therapy model. Both models showed statistically significant graded survival outcome. CONCLUSION:Post-therapy (C2AD2) and pre-therapy (C2AD) models are simple, easy to use and effective tools to stratify breast and lung cancer patients undergoing chest radiation for post-RT MACCE.

Introduction: Radiation therapy (RT) induced carotid artery disease (RICAD) is a significant contributor to morbidity and mortality of oropharyngeal cancer (OPC) survivors. Pre-existing carotid atherosclerosis is likely a risk factor for accelerated development of RICAD.Its evaluation prior to RT is not currently standard practice. This study aimed to utilize standard of care (SOC) oncologic pre-RT CT scans to quantify carotid calcific atherosclerosis and assess the role of carotid artery calcium score (CaACS) in predicting RICAD and its sequalae. Methods: Patients with OPC treated with RT at a tertiary center during 2016-19 were identified. SOC oncologic pre-RT CT of the neck with contrast was used to estimate CaACS utilizing the CT Cardiac package of SyngoVia (Siemens Healthcare). To separate calcified plaque from contrast, density threshold of 230 HU was used.Bilateral common and internal carotid arteries were evaluated to the C1 vertebra. Patients were split into three groups based on CaACS distribution:low (values ≤ median) as reference group,medium(values between median and 75th percentile),and high (values >75th percentile). Results: A total of 271 patients were included (mean age 61 ± 9.8, 88% male).Smoking was reported in 57%, hypertension (HTN) in 53%, dyslipidemia (DLD) in 43%, and diabetes mellitus (DM) in 14%,while 34% were on a statin prior to RT. Total median CaACS was 10.8 AU (IQR 0-140.6) with 46% having a score of 0. During a median follow up of 6.4 years (IQR 6.3-6.6), 41% developed new or worsened carotid atherosclerosis, 3.7% carotid artery stenosis >50%, 2.6% TIA or stroke, and 17% died without new or worsened carotid atherosclerosis. In a multivariate cause-specific Cox regression analysis, high CaACS was significantly associated with increased risk of the composite outcome of worsened carotid atherosclerosis, >50% stenosis, TIA or stroke (HR 2.18,95% CI[1.34-3.54]) compared to low CaACS. Hypertension was also associated with the composite outcome (2.26[1.49-3.41]) while no significant associations were noted with age, smoking, DLD, DM, or statin use. Fine-Gray analysis showed similar results. Conclusion: In our cohort of patients with OPC, high CaACS and history of HTN prior to RT, were significantly associated with development of RICAD. Quantification of carotid artery calcific atherosclerosis utilizing SOC oncologic CT imaging may assist with patient risk stratification, guide surveillance and potentially mitigate RICAD in OPC survivors.

Artificial intelligence has revolutionized computational biology. Recent developments in omics technologies, including single-cell RNA sequencing and spatial transcriptomics, provide detailed genomic data alongside tissue histology. However, current computational models focus on either omics or image analysis, lacking their integration. To address this, we developed OmiCLIP, a visual-omics foundation model linking hematoxylin and eosin images and transcriptomics using tissue patches from Visium data. We transformed transcriptomic data into 'sentences' by concatenating top-expressed gene symbols from each patch. We curated a dataset of 2.2 million paired tissue images and transcriptomic data across 32 organs to train OmiCLIP integrating histology and transcriptomics. Building on OmiCLIP, our Loki platform offers five key functions: tissue alignment, annotation via bulk RNA sequencing or marker genes, cell-type decomposition, image-transcriptomics retrieval and spatial transcriptomics gene expression prediction from hematoxylin and eosin-stained images. Compared with 22 state-of-the-art models on 5 simulations, and 19 public and 4 in-house experimental datasets, Loki demonstrated consistent accuracy and robustness.

Background:Atherothrombosis, the main event leading to acute coronary syndrome (ACS), is strongly linked to disturbed blood flow (d-flow) regions. Although the involvement of the Hippo pathway and its kinases Large Tumor Suppressor Kinase 1and 2 (LATS 1 and 2) in mechanical stress responses is known, the mechanisms by which d-flow simultaneously induces senescence, proliferation, and atherothrombosis remain unclear. Methods:The role of endothelial cells (EC)-specific LATS1/2 was examined using EC specific knock-out (EKO) mice in a partial left carotid ligation (PLCL) model. Plaque spatial multi-omics analysis was performed by integrating imaging mass cytometry, sequential immunofluorescence (COMET™), and spatial metabolomics at the single-cell level in human and mouse atherosclerotic plaques. Results:In tamoxifen-inducible Lats1 homo( -/- ) / Lats2 homo( -/- ) EC-specific knockout (EKO) mice, deletion of LATS1/2 induced by tamoxifen led to fatal outcomes, characterized by severe systemic edema and markedly increased vascular permeability. In contrast, Lats1 het(+ /- ) / Lats2 homo( -/- ) -EKO mice survived and developed atherothrombotic plaques exhibiting neovascularization even without further additional dietary or genetic intervention. Spatial proteomics analysis revealed that LATS1/2 depletion in ECs triggered a senescence-associated stemness (SAS) phenotype, primarily driven by CD38 upregulation. Complementary spatial metabolomics profiling demonstrated a significant increase in sulfite and taurine within LATS1/2-deficient plaques, indicating lowered sulfite oxidase (SUOX) activity. Mechanistically, CD38 upregulation was found to suppress SUOX expression, induce the reverse mode of mitochondrial complex V, and increase succinate dehydrogenase (SDH) activity along with ATP consumption. Paradoxically, despite ATP depletion, this metabolic disturbance enhanced glutamate metabolism and the tricarboxylic acid (TCA) cycle, sustaining EC proliferation under energetically stressed conditions. The combined effect of LATS1/2 deletion and CD38 activation established a unique EC phenotype defined by increased SAS, leading to proliferation, senescence, and eventual cell death. These pathological processes culminated in the formation of atherothrombotic plaques, which were attenuated by inhibition of CD38. Notably, a similar phenotype-marked by metabolically active ECs-was observed in human atherothrombotic plaques, suggesting translational relevance. Conclusion:Loss of LATS1/2 in ECs induces SAS state that promotes excessive EC proliferation, senescent cell accumulation, and the development of structurally fragile, leaky neo vessels-hallmarks of atherothrombotic lesions. CD38-mediated SUOX deficiency further amplifies this pathological process by inducing mitochondrial dysfunction, depleting ATP, and triggering compensatory upregulation of glutamate and TCA cycle metabolism. These findings identify a novel LATS1/2-CD38-SUOX axis in ECs that orchestrates SAS-driven atherothrombosis. Targeting CD38 may represent a promising therapeutic strategy to mitigate vascular dysfunction and plaque instability in high-risk ACS patients. Graphical abstract:Under normal physiological conditions, LATS1/2 and Lamin A work together to suppress CD38 expression. Lamin A binds directly to the CD38 promoter to repress transcription, and LATS1/2 interact with Lamin A to reinforce this suppression. This collaboration helps maintain low CD38 activity and preserves cellular NAD⁺ levels. However, under disturbed flow (d-flow), both LATS1/2 and Lamin A are downregulated. The loss of this dual repression leads to increased CD38 NADase expression. Elevated CD38 accelerates NAD⁺ consumption, causing NAD⁺ depletion-a hallmark of cellular senescence. Reduced NAD⁺ disrupts key metabolic and stress-response pathways, contributing to the onset of the senescent state. At the same time, CD38 suppresses sulfite oxidase (SUOX), leading to sulfite accumulation and mitochondrial redox imbalance. This shift activates the reverse mode of mitochondrial Complex V, which decreases ATP production and increases mitochondrial ROS, intensifying metabolic and oxidative stress in endothelial cells (ECs). In response, ECs compensate by upregulating succinate dehydrogenase (SDH), enhancing TCA cycle activity and glutamate metabolism. This metabolic adaptation provides the biosynthetic building blocks needed for cell growth and proliferation. As a result, ECs adopt a paradoxical phenotype: they show classical features of stress-induced senescence (such as NAD⁺ depletion, oxidative stress, and cell cycle arrest signals), while simultaneously undergoing metabolic activation and proliferation, also mediated by YAP. This defines a non-canonical endothelial program known as senescence-associated stemness (SAS), characterized by the formation of abnormal, proliferative, yet fragile neovessels. These dysfunctional vessels contribute to atherothrombosis, setting this process apart from the more stable lesions typical of conventional atherosclerosis.

Significance: This review investigates how radiation therapy (RT) increases the risk of delayed cardiovascular disease (CVD) in cancer survivors. Understanding the mechanisms underlying radiation-induced CVD is essential for developing targeted therapies to mitigate these effects and improve long-term outcomes for patients with cancer. Recent Advances: Recent studies have primarily focused on metabolic alterations induced by irradiation in various cancer cell types. However, there remains a significant knowledge gap regarding the role of chronic metabolic alterations in normal cells, particularly vascular cells, in the progression of CVD after RT. Critical Issues: This review centers on RT-induced metabolic alterations in vascular cells and their contribution to senescence accumulation and chronic inflammation across the vasculature post-RT. We discuss key metabolic pathways, including glycolysis, the tricarboxylic acid cycle, lipid metabolism, glutamine metabolism, and redox metabolism (nicotinamide adenine dinucleotide/Nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADP+)/NADPH). We further explore the roles of regulatory proteins such as p53, adenosine monophosphate-activated protein kinase, and mammalian target of rapamycin in driving these metabolic dysregulations. The review emphasizes the impact of immune-vascular crosstalk mediated by the senescence-associated secretory phenotype, which perpetuates metabolic dysfunction, enhances chronic inflammation, drives senescence accumulation, and causes vascular damage, ultimately contributing to cardiovascular pathogenesis. Future Directions: Future research should prioritize identifying therapeutic targets within these metabolic pathways or the immune-vascular interactions influenced by RT. Correcting metabolic dysfunction and reducing chronic inflammation through targeted therapies could significantly improve cardiovascular outcomes in cancer survivors. Antioxid. Redox Signal. 43, 92-114.

Background: Bidirectional signaling between arteries and adjacent adipose tissue has been implicated in atherosclerosis progression. Adipose tissue characterization by CT imaging techniques has emerged as a novel risk-predicting tool for future cardiovascular events. This study aimed to evaluate whether neck adiposity at the time of cancer diagnosis is associated with the subsequent development of radiation therapy (RT) induced carotid artery disease (RICAD) in patients with head and neck cancer (HNC). Methods: Patients with HNC treated with RT at a tertiary center between 2005 and 2010 were identified. Oncologic CT scans of the neck performed for RT planning purposes were used to quantify adipose tissue and skeletal muscle mass at the C3 spinal level by SliceOmatic, version 5.0 Tomovision (Figure 1). Adipose tissue and skeletal muscle volumes were indexed to account for height. Fine-Gray competing risk regression models with stepwise forward selection were used to evaluate the association between baseline clinical and imaging characteristics with subsequent development of the composite event of new or worsened carotid atherosclerosis, carotid artery stenosis >50%, transient ischemic attack (TIA) or stroke. Results: Of 231 patients, 91% were male and 97% white, with a mean age of 74 ± 9 years. Prior to RT, 42% had hypertension, 28% dyslipidemia, 12% diabetes, and 3.5% prior stroke/TIA. At a median follow-up of 9.8 years (IQR 10.5), 42% developed new or worsened carotid atherosclerosis, 12% carotid stenosis >50%, and 6% stroke/TIA, while 39% expired. In the multivariable Fine-Gray analysis, neck adipose tissue index was significantly associated with the development of the composite event (HR 1.04 per unit increase, 95% CI 1.01–1.06, p = 0.0015). Baseline use of antiplatelet agents was also associated with the composite event (HR 2.18, 95% CI 1.41–3.36, p = 0.0004) while no significant association was noted with other characteristics such as age, traditional cardiovascular risk factors, prior atherosclerotic disease, statin use, RT dose and skeletal muscle mass. Conclusion: Neck adiposity was associated with RICAD in our cohort of HNC patients with long follow up, supporting the hypothesis of an interplay between inflamed perivascular adiposity and development of carotid artery disease post RT in survivors of HNC. Adipose tissue quantification using SOC oncologic CT scans may assist with patient risk stratification and guide surveillance.

The histone H3 lysine 4 (H3K4) methyltransferase KMT2D (also called MLL4) is one of the most frequently mutated epigenetic modifiers in many cancers, including medulloblastoma (MB). Notably, heterozygous KMT2D loss frequently occurs in MB and other cancers. However, its oncogenic role remains largely uncharacterized. Here, we show that heterozygous Kmt2d loss in murine cerebellar regions promotes MB genesis driven by heterozygous loss of the MB-suppressor gene Ptch via the upregulation of tumor-promoting programs (e.g., oxidative phosphorylation [OXPHOS]). Downregulation of the transcription-repressive tumor suppressor NCOR2 by heterozygous Kmt2d loss, along with Ptch+/--increased MYCN, upregulated tumor-promoting genes. Heterozygous Kmt2d loss substantially diminished enhancer marks (H3K4me1 and H3K27ac) and the H3K4me3 signature, including those for Ncor2. Combinatory pharmacological inhibition of the enhancer-decommissioning H3K4 demethylase LSD1 and OXPHOS significantly reduced the tumorigenicity of MB cells bearing heterozygous Kmt2d loss. Our findings suggest the molecular and epigenetic pathogenesis underlying the MB-promoting effect of heterozygous KMT2D loss.

Introduction: Radiation induced carotid artery disease (RICAD) is a major cause of morbidity and mortality among survivors of oropharyngeal cancer. This study leveraged standard-of-care CT scans to detect volumetric changes in the carotid arteries of patients receiving unilateral radiotherapy (RT) for early tonsillar cancer, and to determine dose-response relationship between RT and carotid volume changes, which could serve as an early imaging marker of RICAD. Methods: Disease-free cancer survivors (>3 months since therapy and age >18 years) treated with intensity modulated RT for early (T1-2, N0-2b) tonsillar cancer with pre- and post-therapy contrast-enhanced CT scans available were included. Patients treated with definitive surgery, bilateral RT, or additional RT before the post-RT CT scan were excluded. Pre- and post-treatment CTs were registered to the planning CT and dose grid. Isodose lines from treatment plans were projected onto both scans, facilitating the delineation of carotid artery subvolumes in 5 Gy increments (i.e. received 50-55 Gy, 55-60 Gy, etc.). The percent-change in sub-volumes across each dose range was statistically examined using the Wilcoxon rank-sum test. Results: Among 46 patients analyzed, 72% received RT alone, 24% induction chemotherapy followed by RT, and 4% concurrent chemoradiation. The median interval from RT completion to the latest, post-RT CT scan was 43 months (IQR 32-57). A decrease in the volume of the irradiated carotid artery was observed in 78% of patients, while there was a statistically significant difference in mean %-change (+/- SD) between the total irradiated and spared carotid volumes (7.0 +/- 9.0 vs. +3.5 +/- 7.2, respectively, p<.0001). However, no significant dose-response trend was observed in the carotid artery volume change withing 5 Gy ranges (mean %-changes (+/- SD) for the 50-55, 55-60, 60-65, and 65-70+ Gy ranges [irradiated minus spared]: -13.1 +/- 14.7, -9.8 +/- 14.9, -6.9 +/- 16.2, -11.7 +/- 11.1, respectively). Notably, two patients (4%) had a cerebrovascular accident (CVA), both occurring in patients with a greater decrease in carotid artery volume in the irradiated vs the spared side. Conclusions: Our data show that standard-of-care oncologic surveillance CT scans can effectively detect reductions in carotid volume following RT for oropharyngeal cancer. Changes were equivalent between studied dose ranges, denoting no further dose-response effect beyond 50 Gy. The clinical utility of carotid volume changes for risk stratification and CVA prediction warrants further evaluation. ### Competing Interest Statement EK is supported in part by NIH/NCI 1R01 HL157273 and CPRIT RP200381 both of which are not related to the current work. Dr. Fuller receives related grant, salary, and infrastructure support from MD Anderson Cancer Center via: the Charles and Daneen Stiefel Center for Head and Neck Cancer Oropharyngeal Cancer Research Program; the Program in Image-guided Cancer Therapy; and the NIH/NCI Cancer Center Support Grant (CCSG) Image-Driven Biology and Therapy (IDBT) Program (P30CA016672). Dr. Fuller has received unrelated grants/honoraria from Elekta AB, has received travel support from Philips Medical Systems, and has served in an advisory capacity for Siemens Healthineers. ### Funding Statement This study did not receive any funding ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Institutional Review Board (IRB) of the University of Texas MD Anderson Cancer Center approved this retrospective protocol and waived the need for a consent. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors.

BackgroundTraf2 and Nck-interacting kinase (TNIK) is known for its regulatory role in various processes within cancer cells. However, its role within endothelial cells (ECs) has remained relatively unexplored.MethodsLeveraging RNA-seq data and Ingenuity Pathway Analysis (IPA), we probed the potential impact of TNIK depletion on ECs.ResultsExamination of RNA-seq data uncovered more than 450 Differentially Expressed Genes (DEGs) in TNIK-depleted ECs, displaying a fold change exceeding 2 with a false discovery rate (FDR) below 0.05. IPA analysis unveiled that TNIK depletion leads to the inhibition of the interferon (IFN) pathway [-log (p-value) >11], downregulation of IFN-related genes, and inhibition of Hypercytokinemia/Hyperchemokinemia [-log (p-value) >8]. The validation process encompassed qRT-PCR to evaluate mRNA expression of crucial IFN-related genes, immunoblotting to gauge STAT1 and STAT2 protein levels, and ELISA for the quantification of IFN and cytokine secretion in siTNIK-depleted ECs. These assessments consistently revealed substantial reductions upon TNIK depletion. When transducing HUVECs with replication incompetent E1-E4 deleted adenovirus expressing green fluorescent protein (Ad-GFP), it was demonstrated that TNIK depletion did not affect the uptake of Ad-GFP. Nonetheless, TNIK depletion induced cytopathic effects (CPE) in ECs transduced with wild-type human adenovirus serotype 5 (Ad-WT).SummaryOur findings suggest that TNIK plays a crucial role in regulating the EC response to virus infections through modulation of the IFN pathway.

Background: While the shelterin complex, including TERF2IP, is established in its role of telomere protection, its interaction with the Hippo pathway, which also regulates senescence and inflammation, has not been reported. Our research aims to investigate whether TERF2IP influences the Hippo pathway, potentially linking it to endothelial cell (EC) responses under disturbed flow (d-flow). Methods: ECs were transduced with TERF2IP variants, and subjected to d-flow or static conditions. Post-transfection with makron-1 ubiquitin E3 ligase (MKRN1)/control siRNA, ECs underwent d-flow or remained static. Protein turnover was analyzed using cycloheximide (CHX) treatment. TERF2IP-MKRN1/LATS1/2 interactions were assessed via co-immunoprecipitation after d-flow exposure. Results: LATS1/2 depletion led to endothelial cell (EC) senescence and apoptosis, prompting investigation into the regulatory role of TERF2IP S205 phosphorylation on d-flow-induced LATS1/2 degradation. Adenoviral transduction with the TERF2IP S205A mutation significantly mitigated the d-flow-mediated decrease in LATS1/2 levels. The absence of changes in LATS1/2 mRNA post-d-flow and the protective effect of the protease inhibitor MG132 against LATS1/2 reduction implicate protein degradation mechanisms. Furthermore, MKRN1 depletion markedly curtailed the d-flow-induced decline in LATS1/2 and senescence, underscoring the involvement of the MKRN1 Ub E3 ligase in regulating LATS1/2 protein stability and d-flow-mediated EC senescence. CHX runoff assays following MKRN1 or control siRNA transfection revealed that MKRN1 is pivotal for LATS1/2 stability. The interaction between TERF2IP and MKRN1, enhanced by d-flow and attenuated by the TERF2IP S205A mutation, along with the inhibitory effect of TERF2IP c-myb domain overexpression on their association, indicates a binding site for MKRN1 on TERF2IP. Persistent TERF2IP-LATS1/2 binding until LATS1/2 expression declined suggests the formation of a TERF2IP-MKRN1-LATS1/2 complex that facilitates LATS1/2 degradation mediated by the recruitment of TERF2IP-MKRN1 complex. Conclusion: D-flow triggers the phosphorylation of TERF2IP at S205, leading to the formation of a TERF2IP-MKRN1-LATS1/2 complex. This complex initiates the degradation of LATS1/2, a key regulator of EC senescence. Thus, TERF2IP S205 phosphorylation under d-flow conditions is a crucial step in the pathway leading to LATS1/2 proteolysis, impacting our understanding of EC senescence and atherothrombosis.

Background: D-flow’s impact on metabolic reprogramming, particularly glycolysis, needs reevaluation due to its conflicting role in atherosclerosis. We show that d-flow reduces LATS1/2 expression, leading to both EC proliferation and senescence. However, the exact molecular mechanisms remain elusive. Methods: We utilized EC-specific Lats1 +/- /Lats2 -/- knockout (LATS1/2-EKO) mice in a partial left carotid ligation (PLCL) model under hypercholesterolemia with AAV-PCSK9 injection to mimic d-flow. Anti-CD38 antibody (Ab68) and IgG2a control were administered intraperitoneally every 5 days at a dose of 5 mg/kg starting one day before PLCL. Plaque characterization was performed using imaging mass cytometry and sequential immunofluorescence (COMET TM ), integrating spatial metabolite data from Bruker timsTOF Flex Mass Spectrometer, and spatial single-cell metabolite data via the Visiopharm platform. Results: EC LATS1/2 depletion induced a unique atherothrombosis lesion with induced EC proliferation and senescence-associated secretory phenotype (SASP). Utilizing spatial single-cell metabolomics, we cataloged 134 distinct metabolites within the ECs. Subsequent overrepresentation analysis revealed that pathways related to glutamate and the citric acid cycle were enhanced in LATS1/2-EKO mice. Specifically, CD38-dependent downregulation of the pyruvate dehydrogenase (PDH) substrate lipoamide decreased PDH enzymatic activity, linking to CD38 to senescence induction. This inhibition led to a metabolic shift towards YAP-dependent glutaminolysis, and upregulated the citric acid cycle. The use of a glutaminase 1 inhibitor (CB-839) significantly curtailed senescence-associated stemness (SAS), underscoring the pivotal role of CD38 in driving glutaminolysis that contributes to d-flow-induced SAS. Moreover, the application of Ab68 but not IgG2a control decreased atherothrombosis formation following PLCL in LATS1/2-EKO mice. However, the effectiveness of Ab68 in attenuating atherosclerosis in wild-type mice was less pronounced. Conclusions: In the presence of d-flow, upregulated CD38, resulting from reduced LATS1/2 expression, triggers EC responses characterized by increased glutaminolysis and the emergence of an EC SAS phenotype. These processes play a crucial role in a unique form of atherothrombosis. Additionally, CD38’s impact on reducing PDH activity, along with YAP and CD38-mediated enhancement of glutaminolysis, constitutes critical pathways for inducing EC SAS by d-flow.

B-1 cells, an innate-immune-like B-cell subset, mainly develop from fetal progenitors. B-1 cells secrete natural IgM antibodies (NAbs), which bind Oxidized-LDL (ox-LDL) that is expressed in the atherosclerotic lesion and apoptotic cells. Therefore, NAbs secreted by B-1 cells clear apoptotic cells and cell debris and play key roles in preventing atherosclerosis (ASC) and chronic inflammation caused by aging (Inflammaging). While several investigations showed the atheroprotective effect of B-cells in the standard atherosclerotic model, it is unclear whether B-1 cells can reduce atherosclerosis in the clonal hematopoiesis-related atherosclerosis model. Ldlr-null mice transplanted with clonal hematopoiesis (CH) bone marrow cells (Tet2-KO mouse BM) on high-fat diets are an advanced model that reflects the status of human patients with severe atherosclerosis and CH (CH-ASC model). It has been reported that CH is observed in at least 10% of people older than 70 years and increases the risk of atherosclerotic cardiovascular diseases. We hypothesize that fetal-derived young B-1 cells prevent the progression of atherosclerosis in CH mice by secreting NAbs that reduce chronic inflammation and that aging of B-1 cells may alter their function. We repeatedly transferred sorted peritoneal cavity (PerC) B-1 cells into CH-ASC model mice with high-fat diets and evaluated atherosclerotic regions as well as immune profiles. The area of aortic plaque formation measured by oil-red staining was significantly reduced in the CH-ASC + B-1 therapeutic transfer group (CH-ASC+B1) compared to CH-ASC alone (30.8 ± 4.7% vs 18.3 ± 6.7%, p<0.03). Plaque formation in the aortic valve also exhibited a reduction in the CH-ASC+B1 group (234.8 ± 37.1 vs 360.3 ± 62.2 sqm, p<0.05). PerC Macrophage (Mac) in the CH-ASC+B1 showed marked increase of M2 polarization (3.2 ± 0.35 x10e5 vs 0.47 ± 0.10 x 10e5, p< 0.01). Plasma anti-PC IgM Abs were measured by ELISA, showing that CH-ASC+B1 plasma before the evaluation had significantly higher than that of CH-ASC (2821 ± 1988 vs 654 ± 611). Interestingly, PerC from CH-ASC showed a dramatic increase of Tet2-KO-derived B-1a cells that are not normally seen in the post-transplant recipient. However, B-1a cells from CH-ASC+B1 PerC had normal frequency of B-1a cells (41.2 ± 6.2 % vs 19.8 ± 9.7 %, p<0.05), suggesting that Tet2-null B-1a cells may not have atheroprotective property. In addition to the secretion of anti-PC IgM, we explored the impact of B-1a cells on M1/M2 polarization. We sorted B-1a cells and Mac from the PerC and stimulated Mac by Interferon-γ + LPS (Mac-I/L) with or w/o the presence of B-1a cells. Twenty-four hours after stimulation, B-1a and Mac were sorted, and cytokine profiles were measured by qPCR. TNF-α and IL-1β were markedly increased in Mac-I/L, whereas Mac-I/L with B-1a demonstrated restoration down to M0 level, indicating that B-1a cells can suppress Mac activation by cell-to-cell mediated function. Taken together, our data demonstrated that young B-1 cell injection successfully reduced atherosclerotic lesions in CH mice and that the ability of anti-PC IgM Ab secretion was reduced in CH mice. Also, our data indicates the critical role of fetal-derived B-1 cells well-functioning in adult mice and the possibility that the alteration of B-1 cell function causes age-associated diseases.

Background: Atherogenesis often develops in regions with disturbed blood flow (d-flow), involving increased ERK5 and p53 SUMOylation due to SENP2 T368 phosphorylation, leading to increased ERK5 and p53 SUMOylation, contributing to endothelial cell (EC) activation. Conversely, laminar flow (l-flow) induces SENP2 S344 phosphorylation, reducing ERK5 and p53 SUMOylation, thereby suppressing EC activation. The presence of senescent ECs in atherosclerotic plaques suggests that d-flow-induced EC senescence may contribute to atherogenesis, possibly due to metabolic changes. EC glycolysis is crucial for NO production and atheroprotection, however, it is inhibited by l-flow, indicating that l-flow protective effects extend beyond glycolysis. Methods: In ECs from wild-type mice exposed to l-flow, the expression of DDIAS increases, regulated by SENP2 S344 phosphorylation. DDIAS interacts with ACLY, a key regulator of fatty acid metabolism and vascular function. We performed LC-MS and IC-MS analyses on ECs with DDIAS knocked down or control siRNA exposed to l-flow, using 13 C 2 -gucose or 13 C 5 -glutamine to trace metabolic changes. HMG-CoA, acetyl-CoA, and CoQ levels were measured using LC-MS and triple quadrupole LC-MS/MS. Results: L-flow enhances glycolysis and glutaminolysis, significantly increasing TCA cycle intermediates through upregulated glutamine pathways. DDIAS expression and its interaction with ACLY are also increased by l-flow. Without DDIAS, the ACLY-mediated mevalonate pathway is inhibited, reducing l-flow-induced antioxidant mechanisms. Thus, the DDIAS-ACLY complex is crucial for the antioxidant response triggered by L-flow, which is essential for supporting angiogenesis after hindlimb ischemia. L-flow-induced increases in HMG-CoA and CoQ are inhibited by DDIAS depletion, highlighting the importance of DDIAS in maintaining the balance of metabolic and antioxidant pathways under L-flow. We are investigating the role of DDIAS-ACLY complex on l-flow-mediated upregulation of glycolysis and glutaminolysis. Conclusion: DDIAS plays a key role in mediating l-flow atheroprotective effect by directly interacting with and activating ACLY, essential for the mevalonate pathway and subsequent HMG-CoA and CoQ biosynthesis. Under l-flow conditions, ECs preferentially utilize glutamine over glucose for TCA cycle intermediates, underscoring the importance of the DDIAS-ACLY complex in supporting EC protection and function through enhanced metabolic pathways.

Background: Neovascularization in response to ischemia relies on reactive oxygen species (ROS), inflammatory, and angiogenic signaling in endothelial cells (ECs). Our prior study suggested that the copper (Cu) chaperone Atox1 acts as a Cu-dependent transcription factor for p47phox, facilitating ROS-NFkB-dependent inflammatory responses in ECs stimulated by inflammatory cytokine TNFα. However, the precise mechanism of Atox1’s nuclear translocation during inflammatory angiogenesis is unknown. Posttranslational SUMOylation and deSUMOylation by SENP1 in the cytosol and nucleus play crucial roles in regulating gene expression, protein subcellular localization and signal transduction. In silico analysis identified a conserved potential SUMOylation motif at Lys(K3) of Atox1. Results: Here we show that TNFα or hypoxia stimulation in human aortic ECs, as well as mouse hindlimb ischemia model that promotes inflammatory angiogenesis, significantly increased Atox1-K3 SUMOylation. Mechanistically, ROS induced by TNFα or hypoxia led to the oxidation/inactivation of SENP1 at Cys603 in the cytosol, thereby elevating Atox1 K3-SUMOylation (3.6-fold). Unexpectedly, Atox1siRNA or overexpression of “SUMO-dead” Atox1K3R mutant in ECs inhibited TNFα or hypoxia-induced, activation of NFkB (increased cytosolic p-NFkB and its nuclear translocation) as well as Cu-dependent Atox1 nuclear translocation. Additionally, in the nucleus under reducing conditions, ChIP and promoter analysis revealed that deSUMOylation of Atox1 by nuclear active SENP1 is required for Atox1’s transcriptional activity on inflammatory and angiogenic genes VCAM/ICAM, IL-8 and RANTES. Functionally, siAtox1 and “SUMO-dead” Atox1K3R mutant reduced inflammatory and angiogenic responses, including monocyte adhesion, migration and capillary tube formation. In vivo, EC-specific Atox1 -/- mice or CRISPR/Cas9-generated “SUMO-dead” Atox1K3R knock-in mutant mice exhibited significantly reduced blood flow recovery, Mac3+ inflammatory cells, and angiogenesis (CD31+ capillary) in hindlimb ischemia and sponge implant inflammatory angiogenesis models. Conclusions: SUMOylation of Atox1 via oxidative inactivation of SENP1 in the cytosol promotes p-NFkB and the nuclear translocation of both Atox1 and NFkB. Within the nucleus, deSUMOylated Atox1 and NFkB synergistically enhance the expression of inflammatory and angiogenic genes in inflamed or hypoxic ECs, thereby driving neovascularization in ischemic cardiovascular diseases.