BACKGROUND:Atherosclerotic plaque inflammation correlates with risk of rupture, causing myocardial infarction. Lower myocardial infarction risk in young women compared with men abates post-menopause, implicating ERs (estrogen receptors). The ERa (ER alpha) is necessary for estrogen effects on atherosclerosis in mouse models, yet the mechanistic role of ERa in plaque inflammation in both sexes remains unclear. METHODS:The role of ERa in driving endothelial cell (EC) adhesion molecule expression and inflammation was studied in vitro in primary human ECs and in vivo in mice. LDLR (low-density lipoprotein receptor)-knockout mice with EC-specific ERa knockout were compared with ERa-intact littermates after 12 weeks of high-fat diet. RESULTS:In both sexes, EC-specific ERa knockout increased plaque inflammation and expression of adhesion molecules, including ICAM1 (intracellular adhesion molecule 1). In vitro, primary human ECs from young women expressed more ERa and less ICAM1 versus age-matched cells from men. ERa knockdown in human coronary ECs from both sexes increased adhesion molecules. Because the MR (mineralocorticoid receptor) has been implicated in ICAM1 expression and plaque inflammation in males, the impact of ERa on MR-induced ICAM1 expression was explored. In human ECs, estrogen prevented aldosterone induction of ICAM1 and MR enrichment on the ICAM1 promoter. In vivo, EC-specific MR-knockout and EC-ERa/MR-double-knockout/LDLR-knockout mice were studied as above. In females, EC-specific MR knockout did not impact ICAM1 or plaque inflammation, consistent with ERa inhibiting MR function. In the double-knockout model, the lack of MR prevented the increased inflammation and ICAM1 expression observed with loss of EC-ERa. In males, EC-specific MR knockout alone decreased inflammation and ICAM1. In the double-knockout model, the proinflammatory effects of MR and the anti-inflammatory impact of ERa offset each other. CONCLUSIONS:These findings reveal a role for ERa in regulating plaque inflammation in both sexes. In females, estrogen acts via EC-ERa to inhibit MR transcriptional upregulation of ICAM1, attenuating plaque inflammation. In males, ICAM1 expression is driven by the MR and inhibited by ERa.

BACKGROUND:Imatinib, the first Abl-tyrosine kinase inhibitor (TKI), improved leukemia outcomes without cardiovascular side effects. Newer agents, including ponatinib, addressed imatinib resistance, improving cancer remission, but substantially increased arterial thrombotic events, including myocardial infarction (MI) and stroke. The mechanism behind ponatinib-induced thrombosis and the cardiovascular effect of asciminib, a newly approved Abl-TKI, remain unknown. METHODS:The effect of clinically relevant plasma concentrations of imatinib, ponatinib, and asciminib were compared with vehicle in vivo using SR-BI-mut/LDLR-knockout (KO) mice to assess spontaneous MI and stroke risk. The mechanism was interrogated in C57BL/6J mice, assessing leukocyte trafficking and thromboinflammation by intravital microscopy and flow cytometry, respectively, and in ApoE-KO mice, assessing plaque phenotype by flow cytometry and histology. In vitro effects on human umbilical vein endothelial cells (ECs) and human coronary artery ECs were determined by flow cytometry, PCR, and immunoblotting. The role of TNF (tumor necrosis factor) signaling was evaluated by pharmacological inhibition and small interfering RNA knockdown. RESULTS:In SR-BI-mut/LDLR-KO mice, ponatinib significantly accelerated death from MI and stroke compared with vehicle, imatinib, and asciminib. In human ECs, only ponatinib increased expression of TNF receptors (TNFRs) and adhesion molecules (P-selectin, ICAM1 [intercellular adhesion molecule 1], and VCAM1 [vascular cell adhesion molecule 1]). Ponatinib rapidly induced TNFR2 membrane trafficking and TNF signaling in human umbilical vein ECs. TNFR inhibition or TNFR2 knockdown prevented ponatinib induction of EC adhesion molecules. In vivo, ponatinib increased mesenteric vessel adhesion molecules, leukocyte rolling and adhesion to vessels, leukocyte and platelet activation, and platelet-leukocyte aggregates. In ApoE-KO mice, ponatinib increased plaque necrotic core and inflammation, consistent with a rupture-prone phenotype. Asciminib-treated mice developed none of these in vitro or in vivo toxicities. In C57BL/6J mice, TNFR inhibition blocked ponatinib-induced mesenteric adhesion molecule expression and leukocyte trafficking, but not platelet-leukocyte aggregation. TNFR blockade prevented ponatinib-induced plaque inflammation in ApoE-KO mice and MI and stroke in SR-BI-mut/LDLR-KO mice. CONCLUSIONS:Ponatinib, a potent anticancer therapy, activates ECs, platelets, and leukocytes, driving plaque inflammation and death from MI and stroke in mice, mirroring clinical cardiotoxicities in patients with cancer. Asciminib did not induce these effects, suggesting it might be a safer option for imatinib-resistant patients with cancer. Inhibition of TNFR-mediated endothelial activation is sufficient to prevent ponatinib-induced major adverse cardiovascular events.

Inhibitors targeting Abl kinase have dramatically improved survival in Philadelphia chromosome-positive leukemias. First-generation imatinib has minimal cardiovascular side effects, whereas newer agents such as dasatinib, ponatinib, and nilotinib are more effective cancer treatments but carry a high risk of arterial thrombosis. The allosteric Abl kinase inhibitor asciminib was recently approved without long-term cardiovascular follow-up. Previous studies reveal disparate effects of dasatinib, ponatinib, and nilotinib on platelets with consistent evidence of endothelial cell (EC) toxicity. Here, we explore prothrombotic endothelial toxicity mechanisms by comparing exposure to vehicle vs clinically relevant concentrations of imatinib, dasatinib, ponatinib, nilotinib, and asciminib on primary human coronary artery ECs (HCAEC) and mouse models of endothelial injury and vascular thrombosis. Dasatinib and ponatinib increased adhesion of human platelets to HCAEC, specifically when ECs, but not platelets, were exposed to drugs. Dasatinib, ponatinib, and nilotinib impaired HCAEC healing in vitro, whereas only nilotinib impaired healing in vivo and increased von Willebrand factor levels in mice. Dasatinib and ponatinib increased early platelet but not fibrin accumulation in the mouse cremaster arteriole laser injury-induced thrombosis model, and only ponatinib increased platelet-leukocyte aggregate formation. Asciminib had no toxic effect in any of these assays, similar to imatinib. These studies reveal novel and distinct mechanisms by which EC damage induced by dasatinib, ponatinib, and nilotinib contributes to a prothrombogenic EC state. The findings suggest the need for distinct side effect prevention strategies and provide preclinical data supporting vascular safety of asciminib, while awaiting long-term vascular safety follow-up results.

Background: Atherosclerosis (athero) inflammation predicts plaque rupture, causing heart attack and stroke. Female protection from heart attack and stroke is lost with age, implicating estrogen, but by unclear mechanism. Our lab showed that the mineralocorticoid receptor (MR) is expressed in endothelial cells (ECs), where it drives plaque inflammation in male mice by inducing the NFkB-regulated adhesion molecule ICAM1. Females had less inflamed plaques, unchanged by EC-MR-knockout (KO). Hypothesis: We hypothesized that EC-estrogen receptor alpha (ER) protects females from plaque inflammation by inhibiting NFkB and EC-MR induction of ICAM1. Methods and Results: Mice with EC specific MR KO (EC-MR-KO), ER KO (EC-ER-KO), or EC KO of both receptors (DKO) were developed and crossed to the LDLR KO background. EC specific gene recombination of MR and/or ER was confirmed by genomic PCR of lung (rich in ECs) and compared to blood, revealing no recombination in leukocytes. Baseline athero risk factors were unchanged by genotype. Male and female mice of all KO lines and floxed Cre- littermate controls were fed high fat diet for 12 weeks resulting in increased body weight and cholesterol with no difference by genotype in athero risk factors. Aortic arches were digested for flow cytometry to quantify plaque inflammation, confirming less inflamed plaques in females versus males. EC-ER-KO increased plaque inflammation, while DKO prevented this increase in both sexes. Compared to controls, ICAM1 protein expression in the descending aorta was decreased by EC-MR-KO only in males. In both sexes, ICAM1 was increased by EC-ER-KO and unchanged by DKO. Females had increased aortic expression of IkB, the inhibitor of NFkB, compared to males and this was prevented by EC-ER-KO. In primary human aortic ECs (HAECs) in vitro , cells from females expressed significantly less ICAM1 protein compared to males. Conclusions: Female atherogenic mice have less inflamed plaques than males and female HAECs express less ICAM1. In vivo , EC-ER protects from plaque inflammation and suppresses ICAM1 in both sexes and IkB in females, and EC-MR is necessary for this atheroprotection. These data support the concept that EC-ER prevents inflammation by inhibiting NFkB and EC-MR-induced ICAM1 expression.

Background: ABL tyrosine kinase inhibitors (TKI) have dramatically improved chronic myeloid leukemia (CML) survival. Due to resistance to the first ABL-TKI imatinib (Ima), most patients are treated with newer agents, including ponatinib (Pon). Although Pon improves cancer remission, the survival benefit is mitigated by a 4-fold increase in arterial thrombosis. The mechanism of Pon-induced arterial thrombosis and the safety of Asciminib (Asc), a newly approved alternative, are not known. CML patients are older and have notably elevated serum TNFa levels. Hypothesis: Since arterial thrombotic events occur when inflamed vascular plaques rupture, we tested the hypothesis that Pon enhances endothelial cell (EC) response to TNFa, promoting EC inflammation and leukocyte trafficking. Methods and Results: In vitro: Human umbilical vein ECs (HUVECs) were treated with Pon, Ima, or Asc at concentrations observed in humans. Adhesion molecules that mediate leukocyte rolling (E- and P-selectin) and adhesion (ICAM1, VCAM1), and TNF receptors (TNFR) 1 and 2 were quantified at 24 hours by qPCR, flow cytometry (FC), and immunoblot. Pon increased TNFR2, P-selectin, VCAM, and ICAM by FC, qPCR, and immunoblot while Ima and Asc had no impact. In primary human coronary artery ECs Pon, but not Asc nor Ima, similarly increased VCAM and P-selectin mRNA. Pretreatment of HUVECs with a nonspecific TNFR inhibitor prevented Pon induction of ICAM1 and P-selectin mRNA. In vivo: Mice were treated with ABL TKIs by oral gavage to achieve drug plasma levels consistent with those in CML patients. After three days, leukocyte trafficking was quantified by intravital microscopy (IVM) in mouse mesenteric vessels. Pon, but not Asc, increased the number of rolling and adherent leukocytes, and increased serum TNFa by ELISA. Treatment with a TNFR inhibitor blocked Pon induced leukocyte trafficking by IVM. Conclusion: Pon, but not Ima nor Asc, induces expression of TNF receptors and adhesion molecules in human ECs in vitro and promotes leukocyte trafficking in vivo. Since inflamed plaques lead to acute arterial thrombosis, this mechanism could contribute to the high risk of arterial thrombosis in Pon-treated cancer patients. If so, Asc may be a safer choice as it does not induce EC inflammation.

Introduction: Tyrosine kinase inhibitors (TKIs) targeting ABL kinase have transformed the treatment of BCR-ABL positive leukemias. First generation imatinib is well tolerated while the newer TKIs, ponatinib and nilotinib, are more effective for leukemia but both substantially increase risk of acute arterial thrombosis, thereby limiting their clinical benefit over imatinib. Asciminib is a new BCR-ABL inhibitor with an unknown safety profile. We previously showed that BCR-ABL inhibitors that increase thrombosis in humans also cause toxicity in endothelial cells (ECs). Hypothesis: We hypothesize that BCR-ABL TKIs that cause EC dysfunction in vitro delay healing at sites of vascular injury in vivo as a potential mechanism of increased risk of thrombosis. Methods: We used the in vitro scratch wound assay and western blot for endothelial nitric oxide synthase (eNOS) phosphorylation in human umbilical vein (HUV)ECs and a mouse carotid artery wire injury model to assess the impact of BCR-ABL TKIs on EC wound healing capacity in vitro and in vivo . Results: ECs were treated with the Cmax concentration in cancer patients, as were mice, as confirmed by serum LC-MS. In vitro , ponatinib and nilotinib significantly decreased HUVEC wound healing compared to both imatinib and asciminib (p<0.001), with no difference between imatinib and asciminib. Ponatinib significantly decreased peNOS compared to both imatinib and nilotinib (<0.05), while asciminib was not significantly different from any of the other TKIs. In vivo , nilotinib significantly decreased carotid EC wound healing compared to imatinib and asciminib (p< 0.05), while ponatinib was not significantly different from any other TKIs. Carotid thrombosis after wire injury was observed in 3/26 mice treated with ponatinib, 5/23 with nilotinib, 1/21 with asciminib, and 0/26 treated with imatinib, with clot-free survival studies ongoing. Conclusions: Ponatinib and nilotinib cause EC toxicity by distinct mechanisms in vitro , which has implications for mitigating their cardiovascular risk. Asciminib causes little vascular toxicity in vitro or in vivo by these assays. Carotid thrombosis developed in some TKI treated mice, suggesting this model can be used to study the link between impaired EC healing and thrombosis.

Kinase inhibitors (KIs) targeting oncogenic molecular pathways have revolutionized cancer therapy. By directly targeting specific tumor-driving kinases, targeted therapies have fewer side effects compared with chemotherapy. Despite the enhanced specificity, cardiovascular side effects have emerged with many targeted cancer therapies that limit long-term outcomes in patients with cancer. Endothelial cells lining all blood vessels are critical to cardiovascular health and are also exposed to circulating levels of systemic anticancer therapies. Both on- and off-target perturbation of signaling pathways from KIs can cause endothelial dysfunction, resulting in cardiovascular toxicity. As such, the endothelium is a potential source, and also a therapeutic target for prevention, of cardiovascular toxicity. In this review, we examine the evidence for KI-induced endothelial cell dysfunction as a mechanism for the cardiovascular toxicities of vascular endothelial growth factor inhibitors, BCR-Abl (breakpoint cluster region-Abelson proto-oncogene) KIs, Bruton tyrosine inhibitors, and emerging information regarding endothelial toxicity of newer classes of KIs.

BCR-ABL tyrosine kinase inhibitors (TKIs) have dramatically improved survival in Philadelphia chromosome-positive leukemias. Newer BCR-ABL TKIs provide superior cancer outcomes but with increased risk of acute arterial thrombosis, which further increases in patients with cardiovascular comorbidities and mitigates survival benefits compared to imatinib. Recent studies implicate endothelial cell (EC) damage in this toxicity by unknown mechanisms with few side-by-side comparisons of multiple TKIs and with no available data on endothelial impact of recently approved TKIs or novels TKIs being tested in clinical trials. To characterize BCR-ABL TKI induced EC dysfunction we exposed primary human umbilical vein ECs in 2D and 3D culture to clinically relevant concentrations of seven BCR-ABL TKIs and quantified their impact on EC scratch-wound healing, viability, inflammation, and permeability mechanisms. Dasatinib, ponatinib, and nilotinib, the TKIs associated with thrombosis in patients, all significantly impaired EC wound healing, survival, and proliferation compared to imatinib, but only dasatinib and ponatinib impaired cell migration and only nilotinib enhanced EC necrosis. Dasatinib and ponatinib increased leukocyte adhesion to ECs with upregulation of adhesion molecule expression in ECs (ICAM1, VCAM1, and P-selectin) and leukocytes (PSGL1). Dasatinib increased permeability and impaired cell junctional integrity in human engineered microvessels, consistent with its unique association with pleural effusions. Of the new agents, bafetinib decreased EC viability and increased microvessel permeability while asciminib and radotinib did not impact any EC function tested. In summary, the vasculotoxic TKIs (dasatinib, ponatinib, nilotinib) cause EC toxicity but with mechanistic differences, supporting the potential need for drug-specific vasculoprotective strategies. Asciminib and radotinib do not induce EC toxicity at clinically relevant concentrations suggesting a better safety profile.

Obesity induced endothelial dysfunction contributes to the vascular inflammation. MicroRNAs (miRNAs) are implicated in the epigenetic regulation of inflammatory pathways. However, relevant miRNAs in the microvasculature, and their relation to chronic inflammation and metabolic dysfunction are poorly understood. Using micro-RNA Seq approach to profile microRNAs from the endothelial cells (ECs) of brown adipose tissue (BAT) of mice with diet-induced obesity (DIO), we identified microRNA- 485-5p (miR-485-5p) as one of the most differentially regulated miRNA. MiR-485-5p was rapidly induced in ECs in response to TNFα, highly enriched in the BAT vascular endothelium of mice with DIO, and was increased by 1.97 fold in human plasma samples with obesity. MiR-485-5p inhibition decreased TNF-α-induced NF-kB-responsive adhesion molecules VCAM-1 (32.6%) and ICAM-1 (34.7%) and phosphorylation of IKBα in ECs in vitro. Gain and loss-of-function studies revealed that neutralization of miR-485-5p potently inhibited leukocyte adhesion to EC monolayers by 20.8% while the overexpression had the opposite effects. Mechanistically, miR-485-5p inhibited the expression of its target gene PCF11 (~40.3%), a polyadenylation factor subunit, an effect that increased VCAM-1 (2.51 fold) and ICAM-1 (1.43 fold) expression as well as leukocyte adhesion to EC monolayers by 18.6% in vitro. Systemic neutralization of miR-485-5p in mice decreased leukocyte adhesion and rolling by 55.1% and 46.7% respectively by intravital microscopy. Finally, systemic delivery of LNA-anti-miR-485-5p inhibitor resulted in improved glucose and insulin tolerance and energy expenditure in mice with DIO while decreasing Ly6Chigh proinflammatory monocytes in blood as measured by FACS. Our findings indicate that miR-485-5p plays a key regulatory role in controlling vascular inflammation in diet-induced obesity, an effect that could be exploited for therapeutic intervention in obesity-induced chronic inflammation. Disclosure F.Bestepe: None. P.F.O'tierney-ginn: None. P.Alcaide: None. I.Z.Jaffe: None. B.Icli: None. R.Pal-ghosh: None. C.Fritsche: None. K.Lakhotiya: None. S.Smolgovsky: None. J.Weston: None. A.Stepanian: None. F.Alvarado: None. P.Catalano: None. Funding American Diabetes Association (1-16-JDF-046 to B.I.); National Institutes of Health (HL149999)

BACKGROUND:Preeclampsia is a syndrome of high blood pressure (BP) with end organ damage in late pregnancy that is associated with high circulating soluble VEGF receptor (sFlt1 [soluble Fms-like tyrosine kinase 1]). Women exposed to preeclampsia have a substantially increased risk of hypertension after pregnancy, but the mechanism remains unknown, leaving a missed interventional opportunity. After preeclampsia, women have enhanced sensitivity to hypertensive stress. Since smooth muscle cell mineralocorticoid receptors (SMC-MR) are activated by hypertensive stimuli, we hypothesized that high sFlt1 exposure in pregnancy induces a postpartum state of enhanced SMC-MR responsiveness.METHODS:Postpartum BP response to high salt intake was studied in women with prior preeclampsia. MR transcriptional activity was assessed in vitro in sFlt1-treated SMC by reporter assays and PCR. Preeclampsia was modeled by transient sFlt1 expression in pregnant mice. Two months post-partum, mice were exposed to high salt and then to AngII (angiotensin II) and BP and vasoconstriction were measured.RESULTS:Women exposed to preeclampsia had significantly enhanced salt sensitivity of BP verses those with a normotensive pregnancy. sFlt1 overexpression during pregnancy in mice induced elevated BP and glomerular endotheliosis, which resolved post-partum. The sFlt1 exposed post-partum mice had significantly increased BP response to 4% salt diet and to AngII infusion. In vitro, SMC-MR transcriptional activity in response to aldosterone or AngII was significantly increased after transient exposure to sFlt1 as was aldosterone-induced expression of AngII type 1 receptor. Post-partum, SMC-MR-KO mice were protected from the enhanced response to hypertensive stimuli after preeclampsia. Mechanistically, preeclampsia mice exposed to postpartum hypertensive stimuli develop enhanced aortic stiffness, microvascular myogenic tone, AngII constriction, and AngII type 1 receptor expression, all of which were prevented in SMC-MR-KO littermates.CONCLUSIONS:These data support that sFlt1-induced vascular injury during preeclampsia produces a persistent state of enhanced sensitivity of SMC-MR to activation. This contributes to postpartum hypertension in response to common stresses and supports testing of MR antagonism to mitigate the increased cardiovascular risk in women after PE.

Objective: Animal models of atherosclerosis are used extensively to interrogate molecular mechanisms in serial fashion. We tested whether a novel systems biology approach to integration of preclinical data identifies novel pathways and regulators in human disease. Approach and Results: Of 716 articles published in ATVB from 1995 to 2019 using the apolipoprotein E knockout mouse to study atherosclerosis, data were extracted from 360 unique studies in which a gene was experimentally perturbed to impact plaque size or composition and analyzed using Ingenuity Pathway Analysis software. TREM1 (triggering receptor expressed on myeloid cells) signaling and LXR/RXR (liver X receptor/retinoid X receptor) activation were identified as the top atherosclerosis-associated pathways in mice (both P <1.93×10 − 4 , TREM1 implicated early and LXR/RXR in late atherogenesis). The top upstream regulatory network in mice (sc-58125, a COX2 inhibitor) linked 64.0% of the genes into a single network. The pathways and networks identified in mice were interrogated by testing for associations between the genetically predicted gene expression of each mouse pathway-identified human homolog with clinical atherosclerosis in a cohort of 88 660 human subjects. Homologous human pathways and networks were significantly enriched for gene-atherosclerosis associations (empirical P <0.01 for TREM1 and LXR/RXR pathways and COX2 network). This included 12(60.0%) TREM1 pathway genes, 15(53.6%) LXR/RXR pathway genes, and 67(49.3%) COX2 network genes. Mouse analyses predicted, and human study validated, the strong association of COX2 expression ( PTGS2 ) with increased likelihood of atherosclerosis (odds ratio, 1.68 per SD of genetically predicted gene expression; P =1.07×10 − 6 ). Conclusions: PRESCIANT (Preclinical Science Integration and Translation) leverages published preclinical investigations to identify high-confidence pathways, networks, and regulators of human disease.

sFlt1-induced PE produces a state of enhanced sensitivity to stimuli that may be mediated by increased activation of SMC-MR. These data support the use of MR antagonists to mitigate the increased risk of cardiovascular disease in women exposed to PE.

Introduction: Animal models are extensively used to interrogate molecular mechanisms of disease. The apolipoprotein E knockout (ApoE-KO) and low-density lipoprotein receptor (LDLR)-KO mouse models are well established preclinical models of atherosclerosis. Hypothesis: We hypothesized that integrated analysis of preclinical data will identify novel signaling pathways and regulatory networks driving atherogenesis. Comparison of findings between models may implicate model-specific mechanisms of atherosclerosis. Methods: Data were extracted from 716 ApoE KO and 422 LDLR KO manuscripts published in ATVB journal from 1995-2020. Extracted data described the impact of an experimental gene perturbation on atherosclerotic phenotypes. Impacts on plaque size and composition were analyzed using Ingenuity Pathway Analysis, separately for each model and as an aggregate dataset. Results: Distinct regulatory pathways and networks were identified across models and in early versus late stages of atherosclerosis. Of top networks in each model, 8/10 were inflammatory and cell growth networks in the ApoE-KO whereas 6/10 were lipid and carbohydrate metabolism networks in the LDLR-KO dataset. Integration of data identified SC-58125, a cyclooxygenase-2 inhibitor, as the top regulatory network that linked 53% (n=168) of genes implicated in atherosclerosis into a single network. Aggregate data from early atherogenesis (<11 wks. high fat diet (HFD)) was enriched in inflammatory functions, whereas late atherosclerosis (14+ wks. HFD) was associated with cell growth and metabolic pathways. Conclusion: Integration of data from two genetic KO mouse models identified distinct gene programs driving atherosclerosis in each model system. Aggregation of data from two models strengthened key findings and identified novel associations. Finally, combining datasets provided greater power to identify distinct gene pathways and networks in early versus late atherosclerosis.

Introduction: Animal models of human diseases are used extensively to interrogate molecular mechanisms in a reductionist fashion. We tested whether aggregation and integration of preclinical data can identify new causative pathways that faithfully model human disease mechanisms. We have termed this novel technique, the Preclinical Science Integration and Translation (PRESCIANT) method. Methods and Results: Data were extracted from 716 manuscripts in the journal Arteriosclerosis, Thrombosis, and Vascular Biology from 1995-2019 using the apolipoprotein E knockout mouse (ApoE-KO) to study atherosclerosis. We identified 360 unique studies in which genes were experimentally perturbed in ApoE-KO mice to impact atherosclerotic plaque size and/or composition. Impacts of the interventions on plaque size, inflammation, and lipid content were analyzed using Ingenuity Pathway Analysis. The top upstream regulatory network (sc-58125, a COX2 inhibitor) linked 37.2% (134) of the genes implicated in atherosclerosis into a single network. Further, Ingenuity Pathway Analysis identified TREM1 signaling, LXR/RXR activation, and renin-angiotensin signaling as the top 3 pathways associated with changes in atherosclerosis parameters. Specifically, early atherogenesis genes were enriched with pathways associated with inflammatory cell migration and infiltration (including TREM1) whereas late atherosclerosis genes were associated with cell metabolism and survival (including LXR/RXR activation). These two pathways were interrogated in a clinical cohort of 88,660 patients by testing for association between genetically predicted expression of the human homologs of mouse pathway genes and a composite phenotype composed of 27 human atherosclerosis diagnoses. There was a significant enrichment (p<0.01 for both pathways) in the number of human homologs associated with atherosclerosis, including 12 (57.2%) genes in the TREM1 pathway and 15 (53.6%) in the LXR/RXR pathway. Conclusion: PRESCIANT can successfully leverage decades of animal investigations to translate results from large-volume singular preclinical studies to make novel causal inferences into human disease.

Growing recognition of persistent cognitive defects associated with electroconvulsive therapy (ECT), a highly effective and commonly used antidepressant treatment, has spurred interest in identifying its mechanism of action to guide development of safer treatment options. However, as repeated seizure activity elicits a bewildering array of electrophysiological and biochemical effects, this goal has remained elusive. We have examined whether deletion of Narp, an immediate early gene induced by electroconvulsive seizures (ECS), blocks its antidepressant efficacy. Based on multiple measures, we infer that Narp knockout mice undergo normal seizure activity in this paradigm, yet fail to display antidepressant-like behavioral effects of ECS. Although Narp deletion does not suppress ECS-induced proliferation in the dentate gyrus, it blocks dendritic outgrowth of immature granule cell neurons in the dentate molecular layer induced by ECS. Taken together, these findings indicate that Narp contributes to the antidepressant action of ECT and implicate the ability of ECS to induce dendritic arborization of differentiating granule cells as a relevant step in eliciting this response.