Lorenzi, Philip L.

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.

Modulating immune function is a critical strategy in cancer and atherosclerosis treatments. For cancer, boosting or maintaining the immune system is crucial to prevent tumor growth. However, in vascular disease, mitigating immune responses can decrease inflammation and slow atherosclerosis progression. Anti-inflammatory therapy, therefore, presents a unique dilemma for cancer survivors: while it may decrease cardiovascular risk, it might also promote cancer growth and metastasis by suppressing the immune response. Senescence presents a potentially targetable solution to this challenge; senescence increases the risk of both cancer therapy resistance and vascular disease. Exercise, notably, shows promise in delaying this premature senescence, potentially improving cancer outcomes and lowering vascular disease risk post-treatment. This review focuses on the long-term impact of cancer therapies on vascular health. We underscore the importance of modulating senescence to balance cancer treatment’s effectiveness and its vascular impact, and we emphasize investigating the role of exercise-mediated suppression of senescence in improving cancer survivorship.

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.

Patients with cholangiocarcinoma have poor clinical outcomes due to late diagnoses, poor prognoses, and limited treatment strategies. To identify drug combinations for this disease, we have conducted a genome-wide CRISPR screen anchored on the bromodomain and extraterminal domain (BET) PROTAC degrader ARV825, from which we identified anticancer synergy when combined with genetic ablation of members of the mTOR pathway. This combination effect was validated using multiple pharmacological BET and mTOR inhibitors, accompanied by increased levels of apoptosis and cell cycle arrest. In a xenograft model, combined BET degradation and mTOR inhibition induced tumor regression. Mechanistically, the 2 inhibitor classes converged on H3K27ac-marked epigenetic suppression of the serine glycine one carbon (SGOC) metabolism pathway, including the key enzymes PHGDH and PSAT1. Knockdown of PSAT1 was sufficient to replicate synergy with single-agent inhibition of either BET or mTOR. Our results tie together epigenetic regulation, metabolism, and apoptosis induction as key therapeutic targets for further exploration in this underserved disease.

Background: Cellular redox control is maintained by generation of reactive oxygen/nitrogen species balanced by activation of antioxidative pathways. Disruption of redox balance leads to oxidative stress, a central causative event in numerous diseases including heart failure. Redox control in the heart exposed to hemodynamic stress, however, remains to be fully elucidated. Methods: Pressure overload was triggered by transverse aortic constriction in mice. Transcriptomic and metabolomic regulations were evaluated by RNA-sequencing and metabolomics, respectively. Stable isotope tracer labeling experiments were conducted to determine metabolic flux in vitro. Neonatal rat ventricular myocytes and H9c2 cells were used to examine molecular mechanisms. Results: We show that production of cardiomyocyte NADPH, a key factor in redox regulation, is decreased in pressure overload-induced heart failure. As a consequence, the level of reduced glutathione is downregulated, a change associated with fibrosis and cardiomyopathy. We report that the pentose phosphate pathway and mitochondrial serine/glycine/folate metabolic signaling, 2 NADPH-generating pathways in the cytosol and mitochondria, respectively, are induced by transverse aortic constriction. We identify ATF4 (activating transcription factor 4) as an upstream transcription factor controlling the expression of multiple enzymes in these 2 pathways. Consistently, joint pathway analysis of transcriptomic and metabolomic data reveal that ATF4 preferably controls oxidative stress and redox-related pathways. Overexpression of ATF4 in neonatal rat ventricular myocytes increases NADPH-producing enzymes‚ whereas silencing of ATF4 decreases their expression. Further, stable isotope tracer experiments reveal that ATF4 overexpression augments metabolic flux within these 2 pathways. In vivo, cardiomyocyte-specific deletion of ATF4 exacerbates cardiomyopathy in the setting of transverse aortic constriction and accelerates heart failure development, attributable, at least in part, to an inability to increase the expression of NADPH-generating enzymes. Conclusions: Our findings reveal that ATF4 plays a critical role in the heart under conditions of hemodynamic stress by governing both cytosolic and mitochondrial production of NADPH.