Cooke, John P.

BACKGROUND: Atherothrombosis, which underlies most acute coronary syndromes and is driven by intraplaque thrombosis, preferentially occurs in regions of disturbed blood flow (d-flow). Although LATS1/2 (large tumor suppressor kinases 1 and 2) are known regulators of endothelial mechanotransduction, the mechanisms by which d-flow connects endothelial senescence, proliferation, and intraplaque thrombosis remain poorly understood. METHODS: We investigated endothelial cell (EC)-specific roles of Lats1/2 using inducible EC-specific knockout mice in a partial carotid ligation model. Spatial multiomics of human and mouse plaques was performed using imaging mass cytometry, COMET sequential immunofluorescence, and spatial metabolomics. RESULTS: Tamoxifen-induced deletion of both Lats1 and Lats2 (homozygous) in ECs caused fatal edema and increased vascular permeability. In contrast, Lats1 het ( ± ) / Lats2 homo ( −/−) -EC-specific knockout mice survived and developed spontaneous atherothrombotic plaques with neovascularization. Spatial proteomics revealed that LATS1/2 loss induced a senescence-associated stemness phenotype driven by CD38 upregulation. Spatial metabolomics showed sulfite and taurine accumulation, indicating SUOX (sulfite oxidase) deficiency. CD38 suppressed SUOX, demonstrated a switch into the reverse mode of mitochondrial complex V, increased succinate dehydrogenase activity, and promoted ATP consumption. Despite ATP depletion, glutamate metabolism and the citric acid cycle flux increased, sustaining EC proliferation under energetic stress. This senescence-associated stemness state promoted both proliferation and senescence, leading to fragile, leaky neovessels and intraplaque thrombotic lesions. Pharmacological CD38 inhibition attenuated these phenotypes. Similar EC states were observed in human plaques. CONCLUSIONS: Loss of Lats1/2 in ECs induces a CD38-associated senescence-associated stemness-like phenotype that promotes intraplaque thrombosis through mitochondrial metabolic reprogramming, including changes consistent with reverse-mode operation of mitochondrial complex V. These findings define a mechanistic link between disturbed flow, endothelial metabolic reprogramming, and intraplaque thrombosis and hemorrhage.

ABSTRACT 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.

Senescence induced by various stresses reprograms cancer cells to acquire senescence-associated (SA) cell growth (SACG), which allows them to escape senescence-induced cell cycle arrest with enhanced growth potential. ERK5 is a dual kinase-transcription factor, containing an N-terminal kinase domain and transactivation domains in the C-terminal half. Recent data have raised questions regarding the functional role of ERK5 kinase inhibitors in cancer and inflammatory diseases. We aimed to investigate how ERK5 reprograms myeloid cells (MC) to the SA secretory phenotype (SASP) and SACG, consequently leading to atherosclerosis (AthS). AthS was inhibited in ERK5 S496A knock-in (KI) mice. Single-cell analysis of p53 and Ki67 with imaging mass cytometry revealed three groups of MCs with different p53 and Ki67 expression patterns in the plaque. One group showed that p53 and Ki67 were co-expressed lineally, suggesting that this group of cells escaped from the growth-suppression effects of p53, which is also known as a senescence marker. The % of ERK5 S496A KI cells in this group was significantly lower than in wild-type (WT). Also, we found an increase of SA β-gal (SAβ-gal) + Ki67 + and 53BP1 + Ki67 + cells in bone narrow-derived macrophage (BMDMs) isolated from hypercholesterolemia (H-chol) WT mice, suggesting H-chol-induced MC SACG. MC SACG were significantly inhibited in ERK5 S496A KI mice, supporting the role of ERK5 S496 phosphorylation on SACG. RNA sequencing analysis identified the ERK5 S496 phosphorylation-dependent AHR induction by H-chol, and AHR depletion significantly inhibited SACG. We also discovered a critical effect of ERK5 S496 phosphorylation on SUMOylation at a novel site of NRF2 (i.e., K518), which inhibited NRF2 transcriptional activity without affecting ERK5 kinase activity and antagonized oxidized LDL (oxLDL)-induced SASP. Specific ERK5 kinase inhibitors (AX15836 and XMD8-92) inhibited oxLDL-induced ERK5 S496 phosphorylation, suggesting that ERK5 S496 phosphorylation was involved at least in part of the effects of these inhibitors. We have discovered a novel mechanism in which ERK5 S496 phosphorylation directly inhibits NRF2 activity by upregulating NRF2 K518 SUMOylation and AHR induction, subsequently inducing SACG and AthS.

Background: ERK5 (extracellular signal-regulated kinase 5) is a dual kinase transcription factor containing an N-terminal kinase domain and a C-terminal transcriptional activation domain. Many ERK5 kinase inhibitors have been developed and tested to treat cancer and inflammatory diseases. However, recent data have raised questions about the role of the catalytic activity of ERK5 in proliferation and inflammation. We aimed to investigate how ERK5 reprograms myeloid cells to the proinflammatory senescent phenotype, subsequently leading to atherosclerosis. Methods: A ERK5 S496A (dephosphorylation mimic) knock in (KI) mouse model was generated using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat–associated 9), and atherosclerosis was characterized by hypercholesterolemia induction. The plaque phenotyping in homozygous ERK5 S496A KI and wild type (WT) mice was studied using imaging mass cytometry. Bone marrow–derived macrophages were isolated from hypercholesterolemic mice and characterized using RNA sequencing and functional in vitro approaches, including senescence, mitochondria reactive oxygen species, and inflammation assays, as well as by metabolic extracellular flux analysis. Results: We show that atherosclerosis was inhibited in ERK5 S496A KI mice. Furthermore, ERK5 S496 phosphorylation mediates both senescence-associated secretory phenotype and senescence-associated stemness by upregulating AHR (aryl hydrocarbon receptor) in plaque and bone marrow–derived macrophages isolated from hypercholesterolemic mice. We also discovered that ERK5 S496 phosphorylation could induce NRF2 (NFE2-related factor 2) SUMOylation at a novel K518 site to inhibit NRF2 transcriptional activity without altering ERK5 catalytic activity and mediates oxidized LDL (low-density lipoprotein)–induced senescence-associated secretory phenotype. Specific ERK5 kinase inhibitors (AX15836 and XMD8-92) also inhibited ERK5 S496 phosphorylation, suggesting the involvement of ERK5 S496 phosphorylation in the anti-inflammatory effects of these ERK5 kinase inhibitors. Conclusions: We discovered a novel mechanism by which the macrophage ERK5-NRF2 axis develops a unique senescence-associated secretory phenotype/stemness phenotype by upregulating AHR to engender atherogenesis. The finding of senescence-associated stemness phenotype provides a molecular explanation to resolve the paradox of senescence in proliferative plaque by permitting myeloid cells to escape the senescence-induced cell cycle arrest during atherosclerosis formation.

The possible association between the membrane-associated guanylate kinase with inverted domain structure-1 (MAGI1) and inflammation has been suggested, but the molecular mechanisms underlying this link, especially during atherogenesis, remain unclear. In endothelial cells (ECs) exposed to disturbed flow (d-flow), p90 ribosomal S6 kinase (p90RSK) bound to MAGI1, causing MAGI1-S741 phosphorylation and sentrin/SUMO-specific protease 2 T368 phosphorylation-mediated MAGI1-K931 deSUMOylation. MAGI1-S741 phosphorylation upregulated EC activation via activating Rap1. MAGI1-K931 deSUMOylation induced both nuclear translocation of p90RSK-MAGI1 and ATF-6-MAGI1 complexes, which accelerated EC activation and apoptosis, respectively. Microarray screening revealed key roles for MAGI1 in the endoplasmic reticulum (ER) stress response. In this context, MAGI1 associated with activating transcription factor 6 (ATF-6). MAGI1 expression was upregulated in ECs and macrophages found in atherosclerotic-prone regions of mouse aortas as well as in the colonic epithelia and ECs of patients with inflammatory bowel disease. Further, reduced MAGI1 expression in Magi1 –/+ mice inhibited d-flow–induced atherogenesis. In sum, EC activation and ER stress–mediated apoptosis are regulated in concert by two different types of MAGI1 posttranslational modifications, elucidating attractive drug targets for chronic inflammatory disease, particularly atherosclerosis.

1. In canine cutaneous veins, local moderate warming (37°-41°C) augments the responsiveness to postjunctional α1-adrenergic stimulation but depresses that to postjunctional α2-adrenergic activation. Moderate cooling (37°-24°C) has the opposite effect. 2. The effects of warming and cooling on α1-adrenergic responses are buffered by a large receptor reserve, allowing for the predominance of alterations in α2-adrenergic responsiveness.

The Society for Vascular Medicine was founded in 1989. During the subsequent 25 years, the Society has grown to approximately 500 members and has achieved international recognition while making important contributions to vascular disease education, clinical vascular medicine and biology research, and patient care. In celebration of the Society’s 25th anniversary, its past and current presidents reflect on the Society’s history, challenges, and achievements, and emphasize the vital role of the SVM in the discipline of vascular medicine.

Intermittent claudication is the most common symptom of peripheral arterial disease (PAD), in part due to an inadequate rise in limb blood flow with exercise. Claudication causes a severe impairment in functional capacity and quality of life in over 3 million Americans. Basic fibroblast growth factor (bFGF) stimulates angiogenesis in vivo and improves limb blood flow in several animal models of hindlimb ischemia. However, the relative safety and efficacy of angiogenic molecules in the treatment of claudication has not been fully evaluated in prospective, blinded clinical trials. In this study, a randomized, double-blind, placebo-controlled, phase II trial of recombinant human bFGF for the treatment of intermittent claudication was performed. bFGF was administered weekly by intravenous infusions of 2 μg/kg for 6 sequential weeks (total dose 12 μg/kg). The primary efficacy endpoint was change in peak walking time (PWT) on a graded exercise treadmill protocol. Secondary efficacy endpoints included changes in functional status as measured by validated questionnaires. The study was stopped prematurely after treatment of the first 24 subjects due to proteinuria in five of the 16 subjects who received systemic bFGF, which exceeded 1000 mg/24 h in four of these five subjects. The small sample size limited evaluation of the predefined efficacy endpoints; however, there was no significant difference between the treatment and control groups for any of the measures of efficacy. In conclusion, intravenous administration of bFGF delivered at low doses weekly for 6 weeks was associated with a high rate of severe proteinuria. It is speculated that bFGF-related proteinuria in this study was primarily related to the systemic route of administration and the frequent dosing schedule. Future clinical trials of bFGF protein should carefully monitor renal function and consider alternative dosing schedules and drug administration routes.