Background and aims: E-cigarettes (E-cigs) are widely used especially among young people, but the effects on vascular stiffening and remodeling are poorly understood. This study evaluates the effects of short-term e-cig exposure on young vs. adult mice and the effects of e-cig derived chemicals present in plasma and urine of exposed animals on cultured endothelial cells (EC) and vascular smooth muscle cells (SMC). Methods C57BL/6J mice of 6- and 14-weeks of age were exposed to unflavored e-cig vapor with and without nicotine and aortic tissue subjected to in- and ex-vivo assessment of stiffness and histological evaluation. Major chemical constituents of e-cig vapor were analyzed in plasma and urine samples, and cultured human aortic ECs and SMCs were treated with these chemicals, including acrolein, formaldehyde and nicotine. Changes in gene and protein expression were quantified, and functional ROS and MMP assays were performed. Results Pulse wave velocity and ex-vivo myography revealed increased stiffness upon treatment with e-cig vapor, and to a greater extent with the inclusion of nicotine. Aortic elastin content in these mice was decreased when compared to room-air controls. In vitro treatment with some of the different chemical compounds present in e-cig vapor led to an increase in endothelial activation markers, and extracellular remodeling proteins. Wire myography showed an endothelium-independent decrease in relaxation in young murine aortas treated with nicotine. Conclusion Our results indicate that even brief exposure to e-cig vapor leads to marked changes in aortic stiffness and vascular remodeling, potentially predisposing for cardiovascular disease conditions, especially when started at an early age.

MiR-501-3p, a small non-coding RNA, has been linked to vascular damage and arterial stiffness, but its role in abdominal aortic aneurysm (AAA) remains unknown. Blood exosomal microRNAs were extracted, and RNA was also extracted from tissue samples, including both the region of maximum aortic dilation and the transition zone between normal and aneurysmal tissue. The low blood miR-501-3p expression group included more AAA patients and greater size than the high group. Multivariate analysis revealed a significant association between the presence of AAA and low blood miR-501-3p (OR = 6.467, p < 0.05). In AAA tissue, miR-501-3p was lower in the maximum point compared to the border point and correlated with larger AAA sizes. All low expression cases at the maximum point had an AAA ≥ 55 mm, whereas only 9.5

E-cigarettes (E-cigs) are widely used especially among young people, but the effects on vascular stiffening and remodeling are poorly understood. This study evaluates the effects of short-term e-cig exposure on young vs. adult mice and the effects of e-cig derived chemicals present in plasma and urine of exposed animals on cultured endothelial cells (EC) and vascular smooth muscle cells (SMC). C57BL/6J mice of 6- and 14-weeks of age were exposed to unflavored e-cig aerosol with and without nicotine (50

Background Given the escalating global burden of cardiovascular diseases (CVDs), atherosclerosis remains a central focus of research. This chronic disorder is characterized by endothelial dysfunction, lipid dysregulation, inflammation, and a complex interplay of genetic factors. Beyond their canonical role in hemostasis, activated platelets may contribute to vascular pathology through the release of platelet-derived small extracellular vesicles (PDsEVs), which carry diverse molecular cargo. In this study, we investigated potential mechanistic roles of PDsEVs in atherosclerosis, employing integrated transcriptomic and proteomic analyses to identify molecular mediators. Methods Blood samples were obtained from patients with atherosclerosis-associated diseases, including abdominal aortic aneurysm (AAA) and carotid artery stenosis (CAS), as well as from healthy controls. Following sample processing, PDsEVs were isolated. PDsEVs identity and purity were verified using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). RNA from PDsEVs was extracted and subjected to RNA sequencing (RNA-seq) with downstream bioinformatic analysis. Myocyte enhancer factor 2B (MEF2B) was overexpressed in human aortic smooth muscle cells (hAoSMCs) via transfection, followed by proteomic analysis. Results NTA and TEM analyses indicated successful isolation of PDsEVs from human blood samples. RNA-seq of PDsEVs revealed MEF2B enrichment in patients with AAA or CAS. Sequential proteomic analysis after overexpression of MEF2B in hAoSMCs indicated an enhanced proliferative response as a potential mechanistic and pathogenically relevant contributor to atherogenesis. Of particular interest was stratifin (SFN), which emerged among the most highly regulated proteins. Conclusion Collectively, our results identify MEF2B, enriched in PDsEVs, as a potential regulator of aberrant hAoSMC proliferation, thereby promoting progression of atherosclerosis.

Objective:Abdominal aortic aneurysm (AAA) is a common and life-threatening vascular disease. Genetic studies have identified numerous risk loci, many potentially encoding plasma proteins. However, the causal effects of plasma proteins on AAAs have not been thoroughly studied. We used genetic causal inference approaches to identify plasma proteins that have a potential causal impact on AAAs. Methods:Causal inference was performed using two-sample Mendelian randomization (MR). For AAAs, we utilized recently published summary statistics from a multi-population genome-wide association meta-analysis including 39,221 individuals with and 1,086,107 individuals without AAAs from 14 cohorts. We used protein quantitative trait loci (protein quantitative trait loci) identified in two large-scale plasma-proteomics studies (deCODE and UKB-PPP) to generate genetic instruments. We tested 2783 plasma proteins for possible causal effects on AAAs using two-sample MR with inverse variance weighting with common sensitivity analyses. Results:MR identified 90 plasma proteins associated with AAAs at a false discovery rate <0.05, with 25 supported by colocalization analysis. Among those supported by both MR and colocalization were proteins such as PCSK9 (odds ratio [OR], 1.3; 95% confidence interval [CI], 1.2-1.4; P < 1e-10), LTBP4 (OR, 3.4; 95% CI, 2.6-4.6; P < 1e-10), and COL6A3 (OR, 0.6; 95% CI, 0.5-0.7; P < 1e-6). Gene Ontology analysis revealed enrichment of proteins (extracellular matrix; OR, 7.8; P < 1e-4), some with maximal mRNA levels in aortic tissue. Bi-directional MR suggested plasma level changes were not caused by liability to AAA itself. Colocalization analysis showed that an aortic expression quantitative trait locus for COL6A3, and a splicing quantitative trait locus for LTBP4 colocalized with their respective plasma pQTLs and AAA signals. Conclusions:Our results highlight proteins and pathways with potential causal effects on AAAs, providing a foundation for future functional experiments. These findings suggest a possible causal pathway whereby genetic variation affecting extracellular matrix proteins expressed in the aortic wall cause their levels to change in blood plasma, influencing development of AAAs.

BACKGROUND:MicroRNAs are post transcriptional modulators of gene expression. We explored the diagnostic and prognostic value of circulating microRNAs in abdominal aortic aneurysm (AAA) disease, for which currently no established circulating biomarker is available. METHODS:We profiled the expression of 754 human microRNAs in plasma from 187 patients with AAA and 190 matched non-diseased controls. To validate, we used two additional AAA patient cohorts, looking at circulating and aortic tissue-derived microRNA expression, and their correlation to AAA disease phenotype, as well as two murine AAA models. RESULTS:We show that among 12 differentially expressed microRNAs, miR-15a and -659 are the most significantly up-regulated in AAA, whereas miR-1183 and -192 are the most significantly down-regulated. miR-15a is upregulated AAA patient tissues, and in plasma from two murine AAA models. In patients from three different cohorts, miR-15a expression levels in plasma, serum and aortic tunica media are significantly correlated with AAA diameter. Through modulation of miR-15a in human aortic smooth muscle cells, we identify several potential target genes of miR-15a known to be down-regulated in human AAA, suggesting its potential involvement in AAA pathology. Inhibition of miR-15a in vivo demonstrates a significant inhibition of murine aortic diameter growth at day 7. CONCLUSIONS:Our findings suggest that miR-15a is a potential biomarker of AAA. Through in vivo studies and based on its target profile, we show that miR-15a is involved in AAA pathogenesis and could help treatment, but also assist in risk-stratification of AAA patients and identify candidates for early AAA repair.

BACKGROUND:Smoking is a well-established risk factor for abdominal aortic aneurysm (AAA). However, the molecular pathways underlying this relationship remain poorly understood. This study aimed to identify circulating protein mediators that may explain the association between smoking and AAA. METHODS:We conducted a network Mendelian randomization study using summary-level data from the largest available genome-wide association studies. Our primary smoking exposure was the lifetime smoking index, with smoking initiation and cigarettes per day included as supplementary traits. The AAA data set comprised 39 221 cases and 1 086 107 controls. Protein data were sourced from 2 large cohorts: UKB-PPP (the UK Biobank Pharma Proteomics Project), where proteins were measured using the Olink platform in 54 219 individuals, and deCODE, where proteins were measured using the SomaScan platform in 35 559 individuals. Two-sample Mendelian randomization was used to estimate the association between smoking and AAA (βtotal) and between smoking and circulating protein levels (β1). Summary data-based Mendelian randomization was then used to assess the association between smoking-related proteins and AAA risk (β2). Mediation pathways were identified based on the directionality of effect estimates, and the corresponding mediation effects were quantified. RESULTS:Genetically proxied smoking traits were consistently associated with an increased risk of AAA. The lifetime smoking index was associated with the levels of 543 out of 5764 unique circulating proteins, with 470 of these associations replicated in supplementary analyses using additional smoking traits and protein sources. Among the smoking-related proteins, genetically proxied levels of 22 were associated with AAA risk. Eight mediation pathways were identified, with ADAMTS15 (a disintegrin and metalloproteinase with thrombospondin motifs 15), IL1RN (interleukin-1 receptor antagonist protein), MMP12 (matrix metalloproteinases 12), PGF (placental growth factor), PCSK9 (proprotein convertase subtilisin/kexin type 9), and UXS1 (UDP-glucuronic acid decarboxylase 1) representing key mediators. CONCLUSIONS:This study identified numerous circulating proteins that are potentially causally linked to smoking, and 8 of these proteins were found to mediate the association between smoking and AAA risk.

Background:Smoking is a well-established risk factor for abdominal aortic aneurysm (AAA). However, the molecular pathways underlying this relationship remain poorly understood. This study aimed to identify circulating protein mediators that may explain the association between smoking and AAA. Methods:We conducted a network Mendelian randomization (MR) study utilizing summary-level data from the largest available genome-wide association studies. Our primary smoking exposure was the lifetime smoking index, with smoking initiation and cigarettes per day included as supplementary traits. The AAA dataset comprised 39,221 cases and 1,086,107 controls. Protein data were sourced from two large cohorts: UKB-PPP, where proteins were measured using the Olink platform in 54,219 individuals, and deCODE, where proteins were measured using the SomaScan platform in 35,559 individuals. Two-sample MR was employed to estimate the association between smoking and AAA (βtotal) and between smoking and circulating protein levels (β1). Summary data-based MR was then used to assess the association between smoking-related proteins and AAA risk (β2). Mediation pathways were identified based on the directionality of effect estimates, and the corresponding mediation effects were quantified. Results:Genetically predicted smoking traits were consistently associated with an increased risk of AAA. The lifetime smoking index was associated with the levels of 543 out of 5,764 unique circulating proteins, with 470 of these associations replicated in supplementary analyses using additional smoking traits and protein sources. Among the smoking-related proteins, genetically predicted levels of 22 were associated with AAA risk. Eight mediation pathways were identified accounting for 42.7% of the total smoking-AAA association and with mediation effects >4% for ADAMTS15, IL1RN, MMP12, PGF, PCSK9, and UXS1. Conclusion:This study identified numerous circulating proteins potentially causally linked to smoking, and eight of these proteins were found to mediate the association between smoking and AAA risk.

Objective: Abdominal aortic aneurysms (AAAs) arise through complex pathogenesis, and good methods of risk stratification have proved elusive. Further, the lack of medical options short of surgery for treatment, and the requirement for dedicated imaging for identification, result in delayed diagnosis and hamper patient outcomes. Application of circulating biomarkers to effectively assess disease presence and predict progression would improve clinical management and support patient well-being. Exploration for suitable circulating biomarkers of AAAs is still very much in process; however, no disease-specific biomarker has yet been established for effective diagnosis and prognosis. This review aims to contribute enhanced tools for utilizing biomarkers for risk stratification and management of AAA disease. Methods: Utilizing MEDLINE/PubMed, we summarize 44 recent publications covering circulating AAA biomarkers. The biomarkers were categorized and tiered into six subgroups by study design, with prospective studies tiered higher than retrospective observational studies. The classification system separately describes a list of post-interventional monitoring biomarkers. Part of the review also deals with recent approaches to identifying potential AAA biomarkers by genetic inference. Results: Forty individual circulating biomarkers, two plasma protein panels (consisting of 9 or 7 proteins), one plasma-multiomic study, and two micro-RNA (miR) panels revealed correlations to AAA disease risk. Among those, many have already been established as biomarkers for other cardiovascular diseases, meaning feasibility has been proven but disease specificity is lacking. Conclusions: Multiple circulating proteins and miRs have been investigated for their utility as AAA-specific diagnostic or prognostic biomarkers. This work may ultimately identify not only novel AAA biomarkers that are specific for cell type, proteins, metabolites, genetic polymorphisms, and miRNA, but permit framing of comprehensive networks of disease-participating molecules. More robust data with higher disease sensitivity and specificity are needed, along with more multi-centered longitudinal clinical studies with large sample sizes.

Background: Cigarette smoking has been identified as an important risk factor for the development of abdominal aortic aneurysm (AAA) and is known to influence levels of blood proteins. However, protein mediators of the smoking-AAA link remain unexplored. Here, we conducted a two-stage Mendelian randomization (MR) study to comprehensively pinpoint possible protein mediators by leveraging large-scale proteomic and genomic data. Methods: We first assessed the associations between genetically predicted smoking traits (lifetime smoking index, smoking initiation and cigarettes per day) and AAA risk (β total ). We then performed a protein-wide MR analysis to explore circulating proteins associated with smoking traits (β 1 ). Among identified proteins, we established their levels associated with AAA risk (β 2 ). According to directionality (β total vs. β 1 ×β 2 ), pathways were constructed, and corresponding mediation proportions were estimated by (β 1 × β 2 )/ β total ×100%. Genetic instruments for smoking traits were selected by filtering variants at P < 5×10 -8 and r 2 <0.001. Cis variants from encoding gene region ± 1Mb were used as instruments for protein. Summary level data on proteins and AAA were obtained from the deCODE study (N=35,559), Fenland study (N=10,708), and AAAgen consortium (37,214 cases), respectively. MR associations were estimated by the multiplicative random effects inverse variance method and summary-data-based MR method were applicable. We performed colocalization analysis for protein-AAA associations. Multiple testing was controlled with False Discovery Rate. Results: Genetically predicted smoking traits were associated with an increased genetic liability to AAA and genetically predicted levels of 48 proteins. Among 48 smoking-associated proteins, genetically predicted levels of 9 proteins were associated with genetic liability to AAA. Three protein pathways were established. Specifically, genetically predicted levels of WFDC2, MMP12, and SMPD1 mediated approximately 10.0 (1.4-18.6) %, 3.6 (1.7-5.5) %, and 2.2 (95% 0.7-3.7) % of the association between genetically predicted lifetime smoking index and genetic liability to AAA. Colocalization evidence was only observed for the SMPD1-AAA association (PH4=0.98). Conclusions: The study revealed a wide effect of cigarette smoking on blood proteins and suggested three potential pathways linking smoking and AAA, including MMP12 which has long been appreciated as important to AAA formation in model systems.

Lower extremity artery disease (LEAD) is caused by a reduction in blood supply, typically due to arterial atherosclerosis. Although revascularization surgical procedures including angioplasty and bypass surgery are available for LEAD patients to treat severe ischemia, non-healing ulcers or necrosis can develop. These complications lead to amputation in about 5–6% of such patients annually. Advanced treatments such as stem cell transplantation are being employed in selected severe cases, although their effectiveness is only about 40% [ 1 Barrett T.J. Macrophages in atherosclerosis regression. Arterioscler. Thromb. Vasc. Biol. 2020; 40: 20-33 Google Scholar ]. Our research laboratory has long sought solutions for this clinical problem, relying on the well-established surgical hind limb ischemia (HLI) model for LEAD.

Background: Smoking and family history are major risk factors for abdominal aortic aneurysm (AAA). Smoking is a powerful modulator of DNA methylation, and nicotine can cause heritable epigenetic alterations in animals and humans. We found that nicotine infusion and e-cigarette (e-cig) vaping augment murine AAA, and that parental nicotine infusion augments model AAA in offspring, altering tissue DNA methylation patterns. Hypothesis: We investigated the effects of maternal e-cig vaping in mice on their offspring’s experimental AAA risk, and transgenerational DNA methylation. Methods: Two treatment arms were employed: 1) pre-fertilization or 2) peri-natal. Female ApoE-/- or C57BL6 mice (F0) were exposed to e-cig nicotine (24 mg/ml) puffs, 9 sec/min, 1 hour/day (or room air). Arm 1: treated 28 days, then mated to untreated controls. Arm 2: mated after 1 week of e-cig, then maintained on treatment until gestation was complete. Aortic tissue of 10-week-old F1 offspring was evaluated using RRBS-Seq DNA methylation analysis. Parallel offspring underwent angiotensin-II infusion (1μg/kg/min; ApoE-/-) or elastase infusion (2U/ml for 5 min at 120 mmHg; C57) to induce AAA. Aneurysm growth was ultrasound tracked over 28 days. Results: Maternal vape exposure altered aortic DNA methylation in offspring, with numerous differentially methylated regions (DMRs; q<0.1). Hundreds of these DMRs were located in the same genes previously identified in offspring of nicotine-infused mice (see Background). Vaping altered offspring aortic methylation patterns in AAA-related genes, including miRNAs. DMR pathway analysis was enriched in transcription factors. Further, many DMRs were located in genes harboring risk loci for human AAA from our recent collaborative publication. Maternal e-cig exposure augmented AAA size in both models, arms, and genders (p<0.05). AAA incidence and mortality were increased in ApoE-/- in both genders and arms. Effects were most significant in males. Conclusions: E-cig nicotine exposure augments experimental AAA growth in multiple models across generations. These effects are accompanied by broad aortic epigenetic changes, including in key AAA modulator genes, with enrichment in transcription factors that target known AAA-related genes.

Background:Abdominal aortic aneurysm (AAA) is a common and life-threatening vascular disease. Genetic studies have identified numerous associated loci, many potentially encoding plasma proteins. However, the causal effects of plasma proteins on AAA have not been thoroughly studied. We used genetic causal inference approaches to identify plasma proteins that have a potential causal impact on AAA. Methods:Causal inference was performed using two-sample Mendelian randomization (MR). For AAA, we utilized recently published summary statistics from a multi-population genome-wide association (GWAS) meta-analysis including 39,221 individuals with, and 1,086,107 individuals without AAA from 14 cohorts. We used protein quantitative trait loci (pQTLs) identified in two large-scale plasma-proteomics studies (deCODE and UKB-PPP) to generate genetic instruments. We tested 2,783 plasma proteins for possible causal effects on AAA using two-sample MR with inverse variance weighting and common sensitivity analyses to evaluate the MR assumptions. Bayesian colocalization and gene ontology (GO) enrichment analyses provided additional insights. Results:MR identified 90 plasma proteins associated with AAA at FDR<0.05, with 25 supported by colocalization analysis. Among those supported by both MR and colocalization were previously experimentally validated proteins such as PCSK9 (OR 1.3; 95%CI 1.2-1.4; P<1e-10), LTBP4 (OR 3.4; 95%CI 2.6-4.6; P<1e-10) and COL6A3 (OR 0.6; 95%CI 0.5-0.7; P<1e-6). GO analysis revealed enrichment of proteins found in extracellular matrix (ECM, OR 7.8; P<1e-4), some with maximal mRNA levels in aortic tissue. Bi-directional MR suggested plasma level changes were not caused by liability to AAA itself. We then investigated whether variants responsible for expression changes in the aorta also influenced plasma levels and AAA risk. Colocalization analysis showed that an aortic expression quantitative trait locus (eQTL) for COL6A3, and a splicing quantitative trait locus (sQTL) for LTBP4 colocalized with their respective plasma pQTLs and AAA signals (posterior probabilities 0.84 and 0.89, respectively). Conclusions:Our results highlight proteins and pathways with potential causal effects on AAA, providing a foundation for future functional experiments. These findings suggest a possible causal pathway whereby genetic variation affecting ECM proteins expressed in the aortic wall cause their levels to change in blood plasma, influencing development of AAA.

Abdominal aortic aneurysm (AAA) is a highly lethal disease with progressive dilatation of the abdominal aorta accompanied by degradation and remodelling of the vessel wall due to chronic inflammation. Platelets play an important role in cardiovascular diseases but their role in AAA is poorly understood. The present study revealed that platelets play a crucial role in promoting AAA through modulation of inflammation and degradation of the ECM. They are responsible for the up-regulation of SPP1 ( osteopontin, OPN) gene expression in macrophages and aortic tissue, which triggers inflammation and remodeling but also platelet adhesion and migration into the abdominal aortic wall and the intraluminal thrombus (ILT). Further, enhanced platelet activation and pro-coagulant activity results in elevated gene expression of various cytokines, Mmp9 and Col1a1 in macrophages and Il-6 and Mmp9 in fibroblasts. Enhanced platelet activation and pro-coagulant activity was also detected in AAA patients. Further, we detected platelets and OPN in the vessel wall and in the ILT of patients who underwent open repair of AAA. Platelet depletion in experimental murine AAA reduced inflammation and ECM remodeling, with reduced elastin fragmentation and aortic diameter expansion. Of note, OPN co-localized with platelets, suggesting a potential role of OPN for the recruitment of platelets into the ILT and the aortic wall. In conclusion, our data strongly supports the potential relevance of anti-platelet therapy to reduce AAA progression and rupture in AAA patients. Translational perspective Abdominal aortic aneurysm (AAA) is a severe cardiovascular disease (CVD) with high mortality. Since the role of platelets is unclear, we explored platelet-mediated processes in the pathogenesis of AAA. Results from platelet depleted mice and patients with AAA revealed that platelets modulate inflammatory and stiffness-related gene expression of macrophages and fibroblasts. Further, platelets induce the release of osteopontin important for the recruitment of platelets to the aortic wall and to the intraluminal thrombus (ILT). Consequently, platelet depletion significantly reduced aneurysm growth. Thus, therapeutic targeting of platelet activation might be crucial for the treatment of patients to reduce AAA formation and progression.

Background The ATP2B1 gene encodes for a calcium pump, which plays a role in removing Ca2+ from cells and maintaining intracellular Ca2+ homeostasis. Reduction of the intracellular Ca2+ concentration in CD4+ T cells is thought to reduce the severity of colitis, while elevation of Ca2+ in CD4+ T cells induces T cell hyperactivity. Our aim was to clarify the role of ATP2B1 in CD4+ T cells and in inflammatory bowel disease development.Methods A murine CD4+ T cell-specific knockout (KO) of ATP2B1 was created using a Cre-loxP system. CD4+ T cells were isolated from thymus, spleen, and blood using fluorescence-activated cell sorting. To quantify messenger RNA levels, quantitative real-time polymerase chain reaction was performed.Results Although the percentages of CD4+ T cells in both KO mouse spleen and blood decreased compared with those of the control samples, both T-bet (a T helper 1 [Th1] activity marker) and GATA3 (a Th2 activity marker) expression levels were further increased in KO mouse blood CD4+ T cells (vs control blood). Diarrhea and colonic wall thickening (with mucosal changes, including crypt distortion) were seen in KO mice but not in control mice. Prior to diarrhea onset, the KO mouse colon length was already noted to be shorter, and the KO mouse stool water and lipid content were higher than that of the control mice. Tumor necrosis factor alpha and gp91 expressions were increased in KO mouse colon.Conclusions Lack of ATP2B1 in CD4+ T cells leads to Th1 and Th2 activation, which contributes to colitis via elevation of tumor necrosis factor alpha and oxidative stress. ATP2B1 deficiency in CD4+ T cells leads to T helper 1/T helper 2 activation, which in turn increases tumor necrosis factor alpha and oxidative stress. These changes contribute to colitis, which is characterized by diarrhea and colonic wall thickening.

Background: Our lab has identified a role for proprotein convertase subtilisin/kexin type9 (Pcsk9) in murine abdominal aortic aneurysm (AAA). Beyond its pathological effects on degradation of LDL-receptors, and enhancement of tissue inflammation via the NF-kB pathway, reports suggest that it alters vascular smooth muscle cell (VSMC) senescence and metabolism. However, the specific cellular mechanisms connecting Pcsk9 to AAA disease (particularly SMC-derived Pcsk9) are still unclear. Hypothesis: Intercellular communication between VSMCs and macrophages via enhanced local Pcsk9 expression drives macrophages towards pro-inflammatory polarization and promotes AAA growth by upregulated aortic wall inflammation. Pcsk9 also enhances mitochondrial metabolism in VSMCs, contributing to cell senescence. Pcsk9 knockdown inhibits these effects. Aims: We explored the impacts of SMC-derived Pcsk9 on aortic inflammation and VSMC behavior, seeking to further understand AAA pathophysiology and provide a basis for re-purposing Pcsk9 inhibitors as a treatment for non-surgical AAA. Methods: Single-cell RNASeq of murine AAA tissue (porcine pancreatic elastase model) utilizing control and Pcsk9-knockout mice (systemic) was performed to examine local changes in aortic wall cell-subtype gene expression. Pcsk9 silenced VSMCs (with siRNA) were co-cultured with macrophages to assess polarization towards pro-inflammatory phenotype and pro-inflammatory gene/protein expression was identified. VSMCs were also treated either with exogenous Pcsk9 or Pcsk9 siRNA in vitro to assess mitochondrial metabolism via Seahorse assay. Results: Pcsk9 knock-out AAA tissue exhibited reductions in pro-inflammatory gene expression profiles in vascular cell sub-populations (compared to wildtype). In vitro, Pcsk9-silenced VSMC-cocultured macrophages presented decreased inflammatory profile assessed by flow cytometry. Further, exogenous Pcsk9-treated-VSMCs displayed metabolic changes compared to control (Mann-Whitney Test). Conclusions: Our data suggest significant mechanistic involvement of Pcsk9 in AAA disease at the cellular level. Absence of Pcsk9 appears to mitigate vascular inflammation and alter VSMC metabolism in ways that may decelerate AAA.

Background: Abdominal aortic aneurysm (AAA) is a common and life-threatening vascular disease. Genetic studies have identified numerous associated loci, many potentially encoding plasma proteins. However, the causal effects of plasma proteins on AAA have not been thoroughly studied. We used genetic causal inference approaches to identify plasma proteins that have a potential causal impact on AAA. Methods: Causal inference was performed using two-sample Mendelian randomization (MR). For AAA, we utilized recently published summary statistics from a multi-population genome-wide association (GWAS) meta-analysis including 39,221 individuals with, and 1,086,107 individuals without, AAA from 14 cohorts. We used protein quantitative trait loci (pQTLs) identified in two large-scale plasma-proteomics studies (deCODE and UKB-PPP) to generate genetic instruments. We tested 2409 plasma proteins for possible causal effects on AAA using two-sample MR with inverse variance weighting and common sensitivity analyses to evaluate the MR assumptions. Bayesian colocalization and gene ontology (GO) enrichment analyses provided additional insights. Results: MR identified 77 plasma proteins whose levels were associated with AAA at FDR<0.05. Of these proteins, 21/77 (27%) were supported by colocalization analysis (posterior probability H4>0.7), indicating that the plasma levels and AAA likely shared causal variants. Among those supported by both MR and colocalization were previously experimentally validated proteins such as PCSK9 (OR 1.3; 95%CI 1.2-1.4; P<1e-10), along with proteins such as LTBP4 (OR 3.4; 95%CI 2.6-4.6; P<1e-10) and COL6A3 (OR 0.6; 95%CI 0.5-0.7; P<1e-6). GO analysis revealed an enrichment of proteins found in extracellular matrix (ECM, OR 7.8; P<1e-4). Gene expression data suggested that some of these ECM proteins have maximal mRNA levels in aortic tissue. Reverse MR for these ECM proteins found no association between genetic liability for AAA and changes in circulating levels of the proteins – suggesting that changes in their plasma levels were not caused by the aneurysm itself. Conclusions: Our results highlight proteins and pathways with potential causal effects on AAA, providing a foundation for future functional experiments. These findings suggest a possible causal pathway whereby genetic variation affecting ECM proteins expressed in the aortic wall cause their levels to change in blood plasma while simultaneously facilitating the formation of AAA.

Significance: Chronic inflammation is a consistent theme in the progression of cardiovascular disease including AAA. Chronic inflammation contributes to aortic wall weakening in AAA by disrupting the ECM and promoting aortic stiffness. Various cell types (fibroblast, macrophages, VSMCs, and circulating platelets) and inflammatory signaling cascades are implicated in aortic wall weakening. Osteopontin (OPN) is a well-known pro-inflammatory cytokine, which is secreted by pro-inflammatory cells and has been found upregulated in AAA patients. However, its mechanistical pathway in disease’ progression and which cells participate in OPN signaling are unclear. Interestingly, literature suggests that activated platelets can drive OPN secretion in proximal inflammatory cells. Therefore, the aim of this project was to determine whether a mechanistic link exists among the activated platelets and OPN production in cells associated with AAA that eventually increases local aortic stiffness and promotes AAA progression. Methods: Human platelets were isolated from donor blood and activated to obtain activated platelet supernatants (APS). M1 macrophages (M1), adventitial fibroblasts (Fib) and vascular smooth-muscle cells (VSMC) were exposed to APS.Full-length OPN and its fragments, generated by thrombin, plasmin and MMP-2 and -9 cleavage, were incubated with the same cell populations as mentioned above. Stiffness-related gene expression and MMP-2 and -9 activity was investigated utilizing qRT-PCR and gelatin zymography, respectively. Results: On exposure to APS, stiffness related gene expression as well as MMPs activity was significantly altered in the vascular cell types. Downstream effects of OPN on aortic stiffness presented changes in OPN treated cells compared to control. Conclusion: Our results indicate that activated platelets contribute to aortic stiffness and induction of vascular inflammation by modulating OPN expression, suggesting a potential role in AAA pathobiology.

Aims Abdominal aortic aneurysm (AAA) is a common cardiovascular disease with a strong correlation to smoking, although underlying mechanisms have been minimally explored. Electronic cigarettes (e-cigs) have gained recent broad popularity and can deliver nicotine at comparable levels to tobacco cigarettes, but effects on AAA development are unknown. Methods and results We evaluated the impact of daily e-cig vaping with nicotine on AAA using two complementary murine models and found that exposure enhanced aneurysm development in both models and genders. E-cigs induced changes in key mediators of AAA development including cytokine chitinase-3-like protein 1 (CHI3L1/Chil1) and its targeting microRNA-24 (miR-24). We show that nicotine triggers inflammatory signalling and reactive oxygen species while modulating miR-24 and CHI3L1/Chil1 in vitro and that Chill is crucial to e-cig-augmented aneurysm formation using a knockout model. Conclusions In conclusion our work shows increased aneurysm formation along with augmented vascular inflammation in response to e-cig exposure with nicotine. Further, we identify Chill as a key mediator in this context. Our data raise concerns regarding the potentially harmful long-term effects of e-cig nicotine vaping. [GRAPHICS] .

Abdominal aortic aneurysm (AAA) is a common disease with substantial heritability. In this study, we performed a genome-wide association meta-analysis from 14 discovery cohorts and uncovered 141 independent associations, including 97 previously unreported loci. A polygenic risk score derived from meta-analysis explained AAA risk beyond clinical risk factors. Genes at AAA risk loci indicate involvement of lipid metabolism, vascular development and remodeling, extracellular matrix dysregulation and inflammation as key mechanisms in AAA pathogenesis. These genes also indicate overlap between the development of AAA and other monogenic aortopathies, particularly via transforming growth factor β signaling. Motivated by the strong evidence for the role of lipid metabolism in AAA, we used Mendelian randomization to establish the central role of nonhigh-density lipoprotein cholesterol in AAA and identified the opportunity for repurposing of proprotein convertase, subtilisin/kexin-type 9 (PCSK9) inhibitors. This was supported by a study demonstrating that PCSK9 loss of function prevented the development of AAA in a preclinical mouse model.