BACKGROUND:Preeclampsia is a hypertensive disorder of pregnancy characterized by systemic endothelial dysfunction. The pathophysiology of preeclampsia remains incompletely understood. This study used human venous endothelial cell (EC) transcriptional profiling to investigate potential novel mechanisms underlying EC dysfunction in preeclampsia.METHODS:Venous ECs were isolated from postpartum patients with severe preeclampsia and those with normotensive pregnancy using a J wire-based technique in the antecubital vein followed by CD144 (vascular endothelial cadherin) magnetic bead isolation. Venous EC transcriptomes were compared between preeclamptic and normotensive individuals. Differentially expressed genes were carried forward for genetic validation using expression quantitative trait loci from the Genotype-Tissue Expression project as exposures for vascular-specific Mendelian randomization. Functional validation of the top candidate was performed in human umbilical vein ECs using gain- and loss-of-function genetic approaches.RESULTS:Seventeen individuals with preeclampsia and 7 normotensive controls were included. Pairwise analysis yielded 14 protein-coding genes nominally differentially expressed in participants with preeclampsia. Mendelian randomization revealed a significant association between higher genetically predicted METAP1 (methionyl aminopeptidase 1) expression in aortic and tibial arterial tissues and greater risk of preeclampsia. METAP1 overexpression in human umbilical vein ECs decreased angiogenesis, with a 66% decrease in tube formation (P=7.9x10-3) and 72% decrease in cell proliferation (P=2.9x10-2). Furthermore, METAP1 overexpression decreased VEGFA expression and increased expression of multiple preeclampsia-related genes, for example, FLT1, INHBA, and IL1B. Conversely, METAP1 knockdown produced opposite effects on tube formation, cell proliferation, and inflammation-related gene expression.CONCLUSIONS:In a cohort of early postpartum individuals, we observed greater METAP1 expression in venous ECs of women with preeclampsia versus normotensive delivery. Mendelian randomization supported a causal relationship between greater vascular METAP1 expression and higher preeclampsia risk, and functional experiments demonstrated antiangiogenic and proinflammatory effects of METAP1 in human ECs consistent with alterations observed in preeclampsia. Ex vivo EC transcriptomics can identify novel mechanisms underlying preeclampsia pathophysiology, with implications for prevention and treatment.

Background: Impaired angiogenic response in peripheral artery disease (PAD) contributes to the progression of tissue ischemia, but methods to evaluate angiogenesis at the tissue level are limited. We describe a novel approach to measure angiogenesis and identify microRNA (miR)-gene pathways utilizing adipose tissue from patients with PAD. Methods: Patients with PAD undergoing infrainguinal bypass surgery or non-PAD control patients undergoing knee replacement surgery were recruited. Subcutaneous adipose tissue was obtained at the time of surgery. In patients with PAD, adipose tissue was taken from both the proximal and the distal ends of the surgical incision. Angiogenic capacity was measured and miR sequencing was performed. Differentially expressed miRs were defined by p < 0.01 and log2 (fold change) > 1 or < -1. The miRs that correlated with angiogenic capacity and their gene targets were identified. Results: Participants with PAD (n = 10) and control participants (n = 5) were recruited. Capillary sprouting was impaired in distal (p = 0.0014) but not proximal (p = 0.12) PAD adipose tissue. In a subset of samples (controls: n = 4, PAD: n = 6), miR sequencing revealed 56 differentially expressed miRs in distal PAD. Six miRs with a correlation to impaired capillary sprouting and related to angiogenesis using Qiagen Ingenuity Pathway Analysis (IPA) software were identified (miRs 144-3p, 15b-5p, 18b-5p, 20b-5p, 454-3p, and 363-3p). These miRs are predicted to target regulators of angiogenesis including vascular endothelial growth factor A (VEGFA), phosphatase and tensin homologue (PTEN), and cyclin-dependent kinase inhibitor 1A (CDKN1A). Conclusion: Evaluation of ischemic adipose tissue represents a novel approach to gain insight into impaired angiogenesis in PAD. Integration with miR sequencing and target analysis has the potential to identify novel pathways of impaired angiogenesis in PAD.

BACKGROUND:Arterial stiffness and endothelial dysfunction are two important features of cardiovascular injury. Arterial stiffness can be measured by Pulse Wave Velocity (PWV) and endothelial dysfunction can be assessed with reactive hyperemia measured by flow-mediated dilation (FMD). Cardio-Ankle Vascular Index (CAVI) is a recently developed method for measuring arterial stiffness. Studies assessing CAVI's association with established tests of arterial stiffness and endothelial function are limited. METHODS:In a cross-sectional study of adults (ages 18-80) with a range of cardiovascular disease risk burden, we measured CAVI by VaSera, tonometry measures of arterial stiffness [carotid-radial PWV (CRPWV), carotid-femoral PWV (CFPWV)], and ultrasound based brachial artery measures of vasodilator function. RESULTS:We enrolled 100 participants with acceptable quality from 93 subjects for primary analysis. The mean value of CAVI measure was 7.7 ± 1.2. There was a significant association between CAVI and CFPWV (r = 0.609, P < 0.001), which remained significant after adjusting for systolic blood pressure while the association of CAVI with CRPWV was more modest. Higher Framingham Risk Score, older age, history of hypertension and diabetes were significantly associated with higher CAVI and CFPWV. There was not any association between CAVI and FMD. Higher CAVI was associated with lower reactive hyperemia, an indicator of vasodilator function in the microvasculature (r = -0.365, P < 0.001). CONCLUSION:Our findings suggest that CAVI relates to both central and peripheral artery stiffness though is not identical to tonometry measures. CAVI associates with microvascular but not conduit artery vasodilator function consistent with the interrelation of large artery stiffness with small vessel dysfunction.

Background: Traditional combustible cigarette (combustible) use induces endothelial dysfunction accelerating cardiovascular disease progression. Young adults frequently use electronic cigarettes (e-cig) that is linked to impaired endothelial cell (EC) function. We aimed to use EC gene expression analysis to explore altered pathways across patterns of tobacco product use. Methods: Healthy adults aged 18-45 who regularly use e-cig (sole or dual), combustible, and non-use controls were with forearm vein EC biopsy and magnetic bead purification. EC total RNA was isolated and profiled on a NextSeq sequencer. Samples with transcript integrity number (TIN) < 10 were removed, genes were filtered with edgeR::filterByExpr, and analyzed with DESeq2. Differentially expressed genes (DEGs) comparing each use group to the non-use group were defined as log 2 fold change >1 and FDR p<0.01. Pathway analysis was done via Gene Ontology database and analyzed using over-representation analysis with log 2 fold change >1 and p<0.05 and selected based on experimental observation. Results: 64 adults were recruited with a mean age 26±7 years and 34% female. After exclusion of samples not meeting TIN-quality standards, 44 samples were available for analysis sole e-cig (N=13), dual use (N=7), combustible (N=10), non-use (N=14). Each use group revealed multiple DEG compared to non-use (Figure): sole e-cig (50 DEG), dual use (18 DEG), combustible (65 DEG) with 28 DEG shared between sole e-cig use and combustible use suggesting similar patterns of altered EC phenotype. Upregulation of 4 genes 1L1B, PTPRO RYR2, and CLEC4D was shared across all three use groups suggestive of inflammatory activation. Pathway analysis in e-cig alone use group showed multiple pathways related to inflammation and immunity enhanced compared to non-use. Conclusions: Young adults who use e-cig along display evidence of enhanced inflammatory gene expression in isolated EC suggestive of vascular inflammatory activation. Patterns of altered gene expression with e-cig use demonstrated both shared and distinct components compared to combustible cigarette use. Further work is needed to determine the components in e-cig that contribute to altered EC phenotype.

Endothelial cell (EC) dysfunction is prevalent in individuals with insulin resistance and diabetes, characterized by diminished phosphorylation of endothelial nitric oxide synthase (p-eNOS) and reduced nitric oxide (NO) production in the endothelium. Despite its common occurrence, the fundamental mechanism underlying EC dysfunction remains incompletely understood. Dysregulation of microRNAs (miRNAs) has been implicated in EC biology and metabolic disorders, yet studies exploring the functional relevance of these miRNAs are limited. In this investigation, venous EC samples were collected from patients with type 2 diabetes mellitus (T2DM) and non-diabetic (ND) individuals (n=20/group). Subsequent miRNA sequencing revealed 37 upregulated and 21 downregulated miRNAs (P<0.05, L2FC>1). Notably, increased expression of miR-409-3p (L2FC>2) emerged as a potential target associated with EC dysfunction. Validation experiments conducted on human aortic EC (HAEC) exposed to high glucose palmitate (HGPAL) confirmed a significant upregulation of miR-409-3p (P<0.001, L2FC>2). In the same experimental setup, insulin-induced p-eNOS was decreased following HGPAL treatment. However, inhibition of miR-409-3p restored p-eNOS levels (18% to 20%), surpassing controls (21%), by enhancing insulin action in EC. Further investigations involving knockdown and overexpression of miR-409-3p demonstrated its negative regulation of p-eNOS in EC. The molecular mechanism uncovered indicates that miR-409-3p modulates p-eNOS signaling through JNK activity, as confirmed by fluorescence microscopy in patient ECs and by western blot in HAECs. In summary, this study identifies an altered miRNA repertoire in EC derived from T2DM patients, suggesting miR-409-3p as a key player in regulating insulin resistance and EC function. Ongoing study looks is delving into the transcriptional targets of miR-409-3p and their functional implications in both T2DM patients and cultured EC. These findings will shed light on the intricate interplay between miRNAs and EC dysfunction, offering potential avenues for therapeutic exploration in the realm of diabetes-associated vascular complications.

Aims Diabetes leads to dysregulated macrophage immunometabolism, contributing to accelerated atherosclerosis progression. Identifying critical factors to restore metabolic alterations and promote resolution of inflammation remains an unmet goal. MicroRNAs orchestrate multiple signalling events in macrophages, yet their therapeutic potential in diabetes-associated atherosclerosis remains unclear.Methods and results miRNA profiling revealed significantly lower miR-369-3p expression in aortic intimal lesions from Ldlr-/- mice on a high-fat sucrose-containing (HFSC) diet for 12 weeks. miR-369-3p was also reduced in peripheral blood mononuclear cells from diabetic patients with coronary artery disease (CAD). Cell-type expression profiling showed miR-369-3p enrichment in aortic macrophages. In vitro, oxLDL treatment reduced miR-369-3p expression in mouse bone marrow-derived macrophages (BMDMs). Metabolic profiling in BMDMs revealed that miR-369-3p overexpression blocked the oxidized low density lipoprotein (oxLDL)-mediated increase in the cellular metabolite succinate and reduced mitochondrial respiration (OXPHOS) and inflammation [Interleukin (lL)-1 beta, TNF-alpha, and IL-6]. Mechanistically, miR-369-3p targeted the succinate receptor (GPR91) and alleviated the oxLDL-induced activation of inflammasome signalling pathways. Therapeutic administration of miR-369-3p mimics in HFSC-fed Ldlr-/- mice reduced GPR91 expression in lesional macrophages and diabetes-accelerated atherosclerosis, evident by a decrease in plaque size and pro-inflammatory Ly6Chi monocytes. RNA-Seq analyses showed more pro-resolving pathways in plaque macrophages from miR-369-3p-treated mice, consistent with an increase in macrophage efferocytosis in lesions. Finally, a GPR91 antagonist attenuated oxLDL-induced inflammation in primary monocytes from human subjects with diabetes.Conclusion These findings establish a therapeutic role for miR-369-3p in halting diabetes-associated atherosclerosis by regulating GPR91 and macrophage succinate metabolism. Graphical Abstract

BACKGROUND: Preeclampsia is a pregnancy-specific hypertensive disorder associated with an imbalance in circulating proangiogenic and antiangiogenic proteins. Preclinical evidence implicates microvascular dysfunction as a potential mediator of preeclampsia-associated cardiovascular risk. METHODS: Women with singleton pregnancies complicated by severe antepartum-onset preeclampsia and a comparator group with normotensive deliveries underwent cardiac positron emission tomography within 4 weeks of delivery. A control group of premenopausal, nonpostpartum women was also included. Myocardial flow reserve, myocardial blood flow, and coronary vascular resistance were compared across groups. sFlt-1 (soluble fms-like tyrosine kinase receptor-1) and PlGF (placental growth factor) were measured at imaging. RESULTS: The primary cohort included 19 women with severe preeclampsia (imaged at a mean of 15.3 days postpartum), 5 with normotensive pregnancy (mean, 14.4 days postpartum), and 13 nonpostpartum female controls. Preeclampsia was associated with lower myocardial flow reserve (β, −0.67 [95% CI, −1.21 to −0.13]; P =0.016), lower stress myocardial blood flow (β, −0.68 [95% CI, −1.07 to −0.29] mL/min per g; P =0.001), and higher stress coronary vascular resistance (β, +12.4 [95% CI, 6.0 to 18.7] mm Hg/mL per min/g; P =0.001) versus nonpostpartum controls. Myocardial flow reserve and coronary vascular resistance after normotensive pregnancy were intermediate between preeclamptic and nonpostpartum groups. Following preeclampsia, myocardial flow reserve was positively associated with time following delivery ( P =0.008). The sFlt-1/PlGF ratio strongly correlated with rest myocardial blood flow ( r =0.71; P <0.001), independent of hemodynamics. CONCLUSIONS: In this exploratory cross-sectional study, we observed reduced coronary microvascular function in the early postpartum period following preeclampsia, suggesting that systemic microvascular dysfunction in preeclampsia involves coronary microcirculation. Further research is needed to establish interventions to mitigate the risk of preeclampsia-associated cardiovascular disease.

Background: Electronic Cigarettes (e-cig) are popular among young adults raising questions about the cardiovascular health impacts. In research settings, acute e-cig use leads to transient heart rate (HR) elevation. We evaluated how e-cig use intensity relates to HR during daily living using a text-messaging program partnered with continuous HR monitoring. Methods: We enrolled healthy young people (age 18-24) sole e-cig users and non-user controls in a text-messaging based program that assessed self-reported e-cig use intensity (vape sessions and puff count) over 5 two-hour time windows spanning awake period over 2 days. HR was collected using Fitbit and aligned to the text-messaging windows. We compared information collected about use intensity (vape sessions and puff number) to HR measures in two models: linear regression associations at a per person level over the total wake time and a repeated measures analysis based on individual time windows accounting for clustering by participant using compound symmetry. Results: In the 78 participants, mean age 21±2 years was similar in e-cig users (N=59, 47% women, 39% Asian, 7% Black, 9% Hispanic) and non-users (N=19, 61% women, 28% Asian, 22% Black, 6% Hispanic). At baseline e-cig users reported high intensity of use: 26±6 days/month, 16±20 vape sessions/day, 6±5 puffs/session. Over the 2-day monitoring period, total vape sessions reported was 23±26 and total puff 77±68 measured per participant and 2.4±4 vape sessions and 8.1±13.3 puffs per 2 hour window. In the per participant analysis, both higher number of vape sessions and puff counts were associated with higher average HR (β=0.08±0.03, P=0.01 and β=0.03±0.01, P=0.003) but not maximum or minimum HR. In the repeated-measures per window analysis, vape sessions and puff counts were associated with higher average HR (β=0.89±0.14, P<0.0001 and β=0.29±0.05, P<0.0001) as well as maximum HR (β=1.1±0.18, P<0.0001 and β=0.33±0.05, P<0.0001) and minimum HR (β=0.70±0.13, P<0.0001 and β=0.20±0.04, P<0.0001), providing temporal evidence linking variability in vaping intensity measures and HR measures. Conclusion: Our findings using an innovative mobile health platform suggest that the intensity of e-cig use is associated with higher HR during daily living in young people. Further investigation is needed to link short term hemodynamic impact of e-cig use to long-term health impacts.

Pod-based electronic (e-) cigarettes more efficiently deliver nicotine using a protonated formulation. The cardiovascular effects associated with these devices are poorly understood. We evaluated whether pod-based e-liquids and their individual components impair endothelial cell function. We isolated endothelial cells from people who are pod users (n = 10), tobacco never users (n = 7), and combustible cigarette users (n = 6). After a structured use, pod users had lower acetylcholine-mediated endothelial nitric oxide synthase (eNOS) activation compared with never users and was similar to levels from combustible cigarette users (overall P = 0.008, P = 0.01 pod vs never; P = 0.96 pod vs combustible cigarette). The effects of pod-based e-cigarettes and their constituents on vascular cell function were further studied in commercially available human aortic endothelial cells (HAECs) incubated with flavored JUUL e-liquids or propylene glycol (PG):vegetable glycerol (VG) at 30:70 ratio with or without 60 mg/mL nicotine salt for 90 min. A progressive increase in cell death with JUUL e-liquid exposure was observed across 0.0001–1% dilutions; PG:VG vehicle with and without nicotine salt induced cell death. A23187-stimulated nitric oxide production was decreased with all JUUL e-liquid flavors, PG:VG and nicotine salt exposures. Aerosols generated by JUUL e-liquid heating similarly decreased stimulated nitric oxide production. Only mint flavored e-liquids increased inflammation and menthol flavored e-liquids enhanced oxidative stress in HAECs. In conclusion, pod e-liquids and their individual components appear to impair endothelial cell function. These findings indicate the potential harm of pod-based devices on endothelial cell function and thus may be relevant to cardiovascular injury in pod type e-cigarette users.

Patients with T2DM display endothelial dysfunction that predicts cardiovascular risk. We have previously demonstrated reduced eNOS activation in endothelial cells (ECs) isolated from patients with T2DM. We sought to identify drivers of EC dysfunction using a combination of proteomic and transcriptomic approaches by measuring: serum O-link cardiovascular panels II and III along with insulin-mediated eNOS phosphorylation and miRNA profiling (NextGen sequencing) in isolated ECs. In 69 patients (37 with T2DM and 32 age- and sex-similar non-T2DM controls) proteomic analysis identified 35 upregulated and 6 downregulated (FDR p<0.05) proteins that included metabolic, vascular and fluid homeostasis, immune and apoptosis related biomarkers. Several biomarkers related to the degree of insulin-mediated eNOS phosphorylation in isolated EC: (renin r= -0.38, p=0.004, chymotrypsin C r= 0.37, p=0.006, paraoxinase 3, r= 0.35, p=0.009, lipoprotein lipase r= 0.34, p=0.01, superoxide dismutase 2 r= 0.31, p=0.02 and adrenomedullin r= -0.27, p=0.049). In a subset of 10 patients (5/group), we confirmed expression of EC-specific miRNA in ECs purified by CD144 microbeads. Comparing patients with T2DM and controls we found 6 upregulated and 7 downregulated miRNAs (FDR P<0.05). Ingenuity Pathway Analysis shows that these miRNAs are involved in the regulation of inflammation, glucose metabolism, stress signaling and cell cycle pathways. Further we found overlap of differentially expressed miRNA with predicted regulation of biomarkers that were altered in T2DM including: miR-4326 and miR-1270 regulate TNFRSF10A and ALCAM, while, miR-4433-5p and miR-342-3p are involved in regulation of superoxide dismutase 2. Our findings demonstrate that an association of circulating biomarkers particularly those that relate to fluid metabolism and oxidative stress with altered endothelial cell signaling in patients with T2DM. Further we have early evidence of altered non-coding miRNA expression in ECs in T2DM that may be related to inflammation and oxidative stress. Ongoing studies are evaluating the functional implications of altered miRNA expression in regulating vascular function in patients with T2DM.

Introduction: Pod-based electronic (e-) cigarettes including JUUL are the most frequently used product type in young adults. Here, we evaluated the acute and chronic impact of pod-based e-cigarettes on vascular health and their relation to volatile organic compound (VOC) exposure. Methods: We performed a short-term observational study of healthy young adults (N = 106) and recruited pod-based e-cigarette users (N = 48), combustible cigarette users (N = 21), and tobacco nonusers (N = 37). Vascular function via flow-mediated dilation and blood pressure (BP) was measured before and after 10-min structured product use of the participant's own product. Urinary VOCs were measured 1 hour after product use. In human aortic endothelial cells (HAECs) in culture exposed to select VOCs, nitric oxide production was measured with DAF-2 indicator. Results: Among pod-based e-cigarette users, 64% were exclusive users including 37% who had never used combustible cigarettes. Pod-based e-cigarette users and combustible cigarette users had higher systolic BP compared to non-users (121±11 mmHg, 121±13 mmHg, 112±10 mmHg, P=0.0004). Structured pod-based e-cigarette use acutely decreased flow-mediated dilation (-3.2±2.7%), raised systolic and diastolic BP (6±8mmHg, 4±5mmHg) and heart rate (5±7bpm), similar to combustible cigarette use (-2.6±2.6%, 9±8mmHg, 6±5mmHg, 6±6bpm P=0.83, 0.3, 0.4, 0.56 vs pod-based), and to a greater extent than nonuse (0.3±4.1%, 0.7±5mmHg, 0.3±3mmHg, -3±4bpm, P=1.0x10-7, 0.002, 0.003, 2.6x10 -7 ). Differences remained in models adjusted for age, sex, and race. The effect of pod-based e-cigarette use was similar in adults who had never used combustible cigarettes. Levels of VOCs acrolein, acrylamide, acrylonitrile, and crotonaldehyde were associated with changes in vascular health measures. Exposure of HAECs in culture to acrolein and acrylamide reduced acetylcholine-stimulated nitric oxide production. Conclusions: Our findings demonstrated that pod-based e-cigarette use had acute and chronic vascular effects in healthy young adults including those who never used combustible cigarettes. Select VOC metabolites were associated with the vascular changes and altered nitric oxide production suggesting relevance to vascular health.

Microvascular dysfunction contributes to adverse clinical outcomes in patients with peripheral artery disease (PAD). Compromised angiogenesis is an important mechanism of limb ischemia that may be a treatment target but is challenging to assess in patients. Selected circulating microRNA (miRNAs) have been implicated in the pathogenesis of PAD. Thus, we sought to characterize angiogenesis potential and identify altered miRNA in patients with PAD. We collected adipose tissue from ischemic limbs of patients with PAD (n=15, age=64±11, 33% women, 66% T2DM, 61% current smoking) at the time of revascularization surgery. Control adipose tissue was procured patients without PAD undergoing joint replacement surgery (n=6, age=58±6, 50% women, 17% T2DM, 33% current smoking). Using matrigel sprouting assay, we observed a significantly lower angiogenesis response over 8 days in adipose tissue samples from PAD patients compared to controls (p=0.008). We performed miRNA profiling using NextSeq (Illumina Inc.) sequencer. miRNA NGS libraries were prepared using QIAseq miRNA Library Kit. Quality controlled NGS libraries were sequenced according to the manufacturer protocol. Our results demonstrated 40 upregulated and 11 downregulated miRNAs (FDR q<0.01, log2 fold change>2). Using bioinformatics approaches to prioritize differentially expressed miRNA predicted to impact pathways relevant to cardiovascular disease, we identified miR205-5p and miR182-5p as top candidate. There were higher levels of miR205-5p (4.8-fold, FDRq=1.5e-10) and miR182-5p (4.1-fold, FDRq=5.4e-16) in adipose tissue from PAD patients compared to controls. We confirmed the differentially expressed levels using qPCR in selected samples. Ingenuity pathway analysis indicates that miR205-5p inhibits VEGF-A, a key regulator of angiogenesis. We observed a trend toward an association of miR205-5p (r=-0.58, p=0.1) and miR182-5p (r=-0.67, p=0.06) and lower angiogenesis response. Taken together our findings demonstrate impaired microvascular angiogenesis response in patients with PAD along with upregulation of selected miRNA predicted to impair angiogenesis pathways. Further work is needed to confirm a functional role of miRNA in the impaired angiogenesis in patients with PAD.

Introduction: The risk of atherosclerotic cardiovascular disease (ASCVD) is increased in people with diabetes. Diabetes (D) is associated with impaired metabolism and functionality of macrophages (Mps), resulting in chronic inflammation. Identifying factors to combat metabolic alterations and promote resolution of inflammation remains an unmet goal. microRNAs (miRs) orchestrate multiple signaling events in Mps and regulate inflammation. Hypothesis: miR-369-3p regulates metabolic reprogramming in Mps during diabetes-associated atherosclerosis (D-AS). Methods and Results: miRNA-seq profiling from aortic intimal lesions of LDLR-/- mice on a high-fat sucrose containing (HFSC) diet revealed miR-369-3p downregulation with D-AS progression. miR-369-3p was also downregulated in PBMCs derived from D and D-associated ASCVD patients. Cell-type profiling showed miR-369-3p enrichment in mouse bone marrow derived Mps (BMDMs). oxLDL-stimulated BMDMs (foam cells, FCs) resulted in marked downregulation of miR-369-3p, increased secretion of IL1-β, and metabolic shift of oxidative phosphorylation (OXPHOS) to glycolysis, while miR-369-3p overexpression (miR-369-3p O/E) favored OXPHOS over glycolysis and reduced IL1-β secretion. Metabolomics revealed succinate, a key immunometabolite was profoundly elevated extracellularly from FCs, while miR-369-3p O/E reversed this phenotype. Extracellular succinate activates its receptor, GPR91. Indeed, GPR91 was upregulated in FCs while miR-369-3p O/E significantly repressed GPR91. 3’UTR luciferase reporter assay confirmed GPR91 as a miR-369-3p target. miR-369-3p O/E dampened GPR91-mediated inflammasome activation and mitochondrial dysfunction in FCs by decreasing p-ERK, p-DRP1, and NLRP3. Therapeutic delivery of miR-369-3p in LDLR-/- mice fed HFSC diet for 12 weeks reduced plaque size by ORO staining and plasma IL1-β. Flow cytometry of aortic Mps revealed that miR-369-3p decreased GPR91 expression by ~50%, along with a decrease in M1 and increase in M2-like-Mps. Conclusions: miR-369-3p confers a pro-resolving role in regulating lipid-driven inflammation and metabolic rewiring in Mps via targeting GPR91 and preventing D-AS plaque progression.

Background: Previous studies demonstrate changes in autonomic function with combustible and earlier generation electronic cigarette (ecig) use. We sought to explore the effects of nicotine salt containing pod-based ecigs on autonomic regulation in young adult regular users by analyzing indices of heart rate variability (HRV). Methods: We enrolled healthy adults (18-45 yo) who were self-reported tobacco nonusers, pod-based ecig users, or combustible cigarette users. Participants were asked to refrain from tobacco product use at least 6 h before the study visit. Indices of HRV: standard deviation of the NN intervals (SDNN) and the root mean square differences of successive NN intervals (RMSSD, measure of parasympathetic function) were measured by Sphygmacor before and 10 minutes after structured use of participants’ own product. Measurements of urinary levels of cotinine, a nicotine metabolite, and 3HPMA, an acrolein metabolite, were made 1 h after product use. Results: In 133 subjects (age 26±5, 49% women), baseline HRV measures were comparable among tobacco non-users (N=39), pod-based ecig users (N=64), and combustible cigarette users (N=30). Analyses adjusted for age and sex demonstrated that acute pod-based ecig use reduced SDNN compared with non-use (-5.9±2.6ms vs 6.5±3.2ms, P=0.004) and similar to the effect of combustible cigarette use (-10.2±3.8ms, P=0.37). Moreover, acute pod-based ecig use reduced RMSSD compared with non-use (-5.6±3.3ms vs 5.4±4.1ms, P=0.04) and similar to the effect of combustible cigarette use (-10.9±4.9ms, P=0.38). Urinary cotinine, reflective of nicotine exposure, was associated with decreases in SDNN and RMSSD (r=-0.33, P=0.001; r=-0.29,P=0.003, respectively) whereas 3HPMA associated with SDNN and showed a trend toward association with RMSSD (r=-0.27, P=0.006; r=-0.18, P=0.06, respectively). Conclusion: Our data demonstrate an association between acute use of pod-based ecigs and altered cardiac autonomic function in association with the level of nicotine and acrolein exposure.

ABSTRACT Background Pod-based electronic cigarettes (e-cigarettes) use that contain nicotine salts is frequent among youth and young adults; thus, we compared the vascular health effects of pod-based e-cigarette use to combustible cigarette use. Methods and Results We performed a two center observational, cross-sectional study of healthy adults recruited from the community (aged 18-45, N=106) in 3 groups: pod-based e-cigarette users (N=48); combustible cigarette users (N=21); and tobacco nonusers (N=37) and assessed the acute (following structured use) and chronic (resting state after 6 hour tobacco abstinence) effects of pod-based e-cigarette use on endothelial function (brachial artery flow-mediated dilation), blood pressure, and heart rate. Among the pod-based e-cigarette users, 64% were exclusive users including 37% who had never used combustible cigarettes. Pod-based e-cigarette users and combustible cigarette users had higher systolic blood pressure compared to non-users (121±11mmHg, 121±13mmHg, 112±10 mmHg, P=0.0004). Structured pod-based e-cigarette use acutely decreased flow-mediated dilation (−3.2±2.7%), raised systolic and diastolic blood pressure (6±8mmHg, 4±5mmHg) and heart rate (5±7bpm), similar to combustible cigarette use (−2.6±2.6%, 9±8mmHg, 6±5mmHg, 6±6bpm P=0.83, 0.3, 0.4, 0.56 vs pod-based), and to a greater extent than nonuse (0.3±4.1%, 0.7±5mmHg, 0.3±3mmHg, -3±4bpm, P=1.0x10 −7 , 0.002, 0.003, 2.6x10 −7 ). Differences remained robust in models adjusted for age, sex, and race. The effect of pod-based cigarette use was similar in adults who had never used combustible cigarettes. Levels of acrolein, acrylamide, acrylonitrile, and crotonaldehyde were associated with the changes in vascular health measures. Conclusions Overall, our findings suggest that pod-based e-cigarette use has acute and chronic vascular effects in healthy young adults including those who never used combustible cigarettes. Select metabolites derived from volatile organic compounds were associated with the vascular changes suggesting relevance to vascular health.

Microvascular dysfunction contributes to adverse clinical outcomes in patients with peripheral artery disease (PAD). Compromised angiogenesis may be a treatment target for limb ischemia but is challenging to assess in patients. Selected circulating miRNAs have been implicated in the pathogenesis of PAD, thus, we seek to identify and find correlation between altered miRNA signatures and angiogenesis potential in patients with PAD. We collected adipose tissue from ischemic limbs of patients with PAD (n=15, age=64±11, 33% women, 66% T2DM, 61% current smoking) at the time of revascularization surgery. Control adipose tissue was procured from patients without PAD undergoing joint replacement surgery (n=6, age=58±6, 50% women, 17% T2DM, 33% current smoking). Endothelial cell sprouting assay demonstrated lower angiogenesis response over 8 days in adipose tissue samples from PAD patients compared to controls (p=0.008). We performed miRNA profiling using NextSeq (Illumina Inc.) sequencer. miRNA NGS libraries were prepared using QIAseq miRNA Library Kit. Quality controlled NGS libraries were sequenced according to the manufacturer protocol. Our results demonstrated 40 upregulated and 11 downregulated miRNAs (FDR q<0.01, log2 fold change>2) in PAD fat tissue compared to controls. Using bioinformatics approaches to prioritize differentially expressed miRNA predicted to impact pathways relevant to cardiovascular disease, we identified miR205-5p and miR182-5p as top candidate. There were higher levels of miR205-5p (4.8-fold, FDRq=1.5e-10) and miR182-5p (4.1-fold, FDRq=5.4e-16) in adipose tissue from PAD patients compared to controls. We confirmed the differentially expressed levels using qPCR in selected samples. Ingenuity pathway analysis indicates that miR205-5p inhibits VEGF-A, a key regulator of angiogenesis. We observed a trend toward an association of miR205-5p (r=-0.58, p=0.1) and miR182-5p (r=-0.67, p=0.06) and lower angiogenesis response. Taken together our findings demonstrate impaired microvascular angiogenesis response in patients with PAD along with upregulation of selected miRNA predicted to impair angiogenesis pathways.

Aims: S-glutathionylation is a reversible oxidative modification of protein cysteines that plays a critical role in redox signaling. Glutaredoxin-1 (Glrx), a glutathione-specific thioltransferase, removes protein S-glutathionylation. Glrx, though a cytosolic protein, can activate a nuclear protein Sirtuin-1 (SirT1) by removing its Sglutathionylation. Glrx ablation causes metabolic abnormalities and promotes controlled cell death and fibrosis in mice. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a key enzyme of glycolysis, is sensitive to oxidative modifications and involved in apoptotic signaling via the SirT1/p53 pathway in the nucleus. We aimed to elucidate the extent to which S-glutathionylation of GAPDH and glutaredoxin-1 contribute to GAPDH/SirT1/ p53 apoptosis pathway. Results: Exposure of HEK 293T cells to hydrogen peroxide (H2O2) caused rapid S-glutathionylation and nuclear translocation of GAPDH. Nuclear GAPDH peaked 10-15 min after the addition of H2O2. Overexpression of Glrx or redox dead mutant GAPDH inhibited S-glutathionylation and nuclear translocation. Nuclear GAPDH formed a protein complex with SirT1 and exchanged S-glutathionylation to SirT1 and inhibited its deacetylase activity. Inactivated SirT1 remained stably bound to acetylated-p53 and initiated apoptotic signaling resulting in cleavage of caspase-3. We observed similar effects in human primary aortic endothelial cells suggesting the GAPDH/ SirT1/p53 pathway as a common apoptotic mechanism. Conclusions: Abundant GAPDH with its highly reactive-cysteine thiolate may function as a cytoplasmic rheostat to sense oxidative stress. S-glutathionylation of GAPDH may relay the signal to the nucleus where GAPDH transglutathionylates nuclear proteins such as SirT1 to initiate apoptosis. Glrx reverses GAPDH S-glutathionylation and prevents its nuclear translocation and cytoplasmic-nuclear redox signaling leading to apoptosis. Our data suggest that trans-glutathionylation is a critical step in apoptotic signaling and a potential mechanism that cytosolic Glrx controls nuclear transcription factors.

Introduction: Pod-based e-cigarettes including JUUL represent the most commonly used product type and more efficiently deliver nicotine; however, their cardiovascular effects are poorly understood. Methods: We evaluated endothelial function (cell viability by TUNEL and nitric oxide bioavailability using DAF-2DA) in human aortic endothelial cells (HAECs) exposed for 90 minutes to one of four JUUL pod flavors (Menthol, Mint, Mango and Virginia Tobacco) or constituents propylene glycol (PG)/vegetable glycerol (VG) at 30:70 ratio with and without nicotine salt, as well as their heated particle fractions separated with a mini-MOUDI impactor. In healthy young adults, we assessed endothelial cell function in nonusers of tobacco products, pod users and combustible cigarette users for eNOS activation. Results: We observed significant increases in cell death with JUUL e-liquid flavors across 0.0001-1% dilutions. PG/VG vehicle alone and with nicotine salt induced cell death to similar levels as the JUUL e-liquids. All JUUL e-liquids at 0.0001% dilution impaired A23187-stimulated nitric oxide production. Exposure to particle fractions of JUUL e-liquids at 0.001% dilution also reduced A23187-stimulated nitric oxide production. Endothelial cells from pod users showed reduced eNOS activation compared with nonusers and similar to reduced levels in combustible cigarette users (figure). Conclusions: Our data suggest endothelial toxicity of pod-based e-liquids and their constituents, including in young adult users.

Electronic cigarette use has especially risen among adolescents and young adults. The aim of this study was to investigate fasting blood glucose and lipid profiles in chronic combustible cigarette and electronic cigarette users. We evaluated participants aged 21 to 45 (n = 525, mean age 31 ± 7 years, 45% women) without established cardiovascular disease or risk factors who were combustible cigarette users (n = 290), electronic cigarette users (n = 131; 65 sole users and 66 dual users), or never users (n = 104). In the first wave of enrollment (2014-2017), electronic cigarette users reported their products as first, second and third generation devices (e-cig users) and were all largely current (i.e., dual) or former (sole) combustible cigarette users, whereas in the second wave of enrollment (2019-2020), electronic cigarette users all reported pod-based device use (pod users) and included more sole users who were never smokers. In multivariable-adjusted analyses comparing to never users, both sole e-cig users and combustible cigarette users had higher glucose and triglycerides and lower high-density lipoprotein (HDL) cholesterol levels. Dual e-cig users showed higher triglycerides and very-low-density lipoprotein cholesterol, and lower HDL cholesterol compared to never users. In contrast, pod users (both sole and dual) had lipid profiles and glucose levels similar to never users. Overall, users of early generation electronic cigarettes display adverse metabolic profiles. In contrast, pod-based electronic cigarette users have similar lipid profiles to never users. Future studies are needed to understand the cumulative effects of electronic cigarette use on cardiometabolic health.

The use of electronic (e-) cigarettes has rapidly risen in youth and young adults. Pod-based e-cigarettes represent a more evolved generation of these devices that are designed to deliver higher levels of nicotine. Despite their popularity, little is known about the cardiovascular toxicity imposed by the use of e-cigarettes. The JUUL brand of pod-based e-cigarettes holds the majority of market shares in the US. The purpose of this study was to evaluate the effects of four common JUUL pod flavors (Menthol, Mint, Mango and Virginia Tobacco), the product constituents propylene glycol (PG)/vegetable glycerol (VG) vehicle at 30:70 ratio and nicotine salt in 30:70 PG/VG on vascular endothelial cell function. We evaluated endothelial function (cell viability by TUNEL and nitric oxide bioavailability using DAF-2DA) in human aortic endothelial cells (HAECs) following a 90-minute exposure to JUUL pods at dilutions of 0.00001% to 1%. Flavored JUUL pod exposure induced at least 15% endothelial cell death at dilutions of 0.0001% for Menthol, 0.1% for Mint, 0.1-1% for Mango, and 0.0001-1% for Virginia Tobacco. Cells exposed to the PG/VG vehicle alone and in the presence of nicotine salt increased cell death to similar levels as the flavored JUUL pod exposures, suggesting that the vehicle may be an important source of toxicity. All flavored JUUL pods at a dilution of 0.0001% impaired A23187-stimulated nitric oxide in HAECs, suggestive of endothelial dysfunction. Our data suggests that exposure of endothelial cells to flavored JUUL pods results in cytotoxicity and impairs nitric oxide bioavailability, which may be relevant to cardiovascular toxicity. Ongoing experiments focus on the evaluation of oxidative stress and interleukin-6 expression in vascular endothelial cells. Future work will include evaluation of the effects of JUUL e-liquid heated fractions to identify fractions and specific chemical constituents that induce endothelial cell toxicity.