Human Artery Endothelial Cells Research Articles

Cytokine and chemokine receptor profiles in adipose tissue vasculature unravel endothelial cell responses in HIV.

Chronic systemic inflammation significantly increases myocardial infarction risk in people living with HIV (PLWH). Endothelial cell dysfunction disrupts vascular homeostasis regulation, increasing the risk of vasoconstriction, inflammation, and thrombosis, contributing to cardiovascular disease. We aimed to characterize endothelial cell (EC) chemokines, cytokine, and chemokine receptors of PLWH, hypothesizing that in our cohort, glucose intolerance contributes to their differential expression implicated in endothelial dysfunction. Using single-cell transcriptomic analysis, we phenotyped chemokine and cytokine receptor expression on arterial ECs, capillary ECs, venous ECs, and vascular smooth muscle cells (VSMCs) in subcutaneous adipose tissue of 59 PLWH with and without glucose intolerance. Our results show that arterial and capillary ECs express significantly higher interferon and tumor necrosis factor (TNF) receptors than venous ECs and VSMCs. Venous ECs exhibited more interleukin (IL)1R1 and ACKR1 receptors, and VSMCs showed significant IL6R expression than arterial and capillary ECs. When stratified by group, arterial ECs from PLWH with glucose intolerance expressed significantly higher IL1R1, IL6R, CXCL12, CCL14, and ICAM2 transcripts than arterial ECs from PLWH without diabetes. Of the different vascular cell types studied, arterial ECs as a proportion of all ECs in adipose tissue were positively correlated with plasma fasting blood glucose. In contrast, venous ECs and VSMCs were positively correlated with plasma IL6. To directly assess the effect of plasma from PLWH on endothelial function, we cultured human arterial ECs (HAECs) in plasma-conditioned media from PLWH and performed bulk RNA sequencing. Plasma from PLWH stimulated ECs with the upregulation of genes that enrich for the oxidative phosphorylation and the TNF-α via NFK-β pathways. In conclusion, ECs in PLWH show heterogeneous cytokine and chemokine receptor expression, and arterial ECs were the most influenced by glucose intolerance. Further research must explicate cytokine and chemokine roles in EC dysfunction and identify biomarkers for disease progression and therapeutic response.

Radiation-Induced Endothelial Ferroptosis Accelerates Atherosclerosis via the DDHD2-Mediated Nrf2/GPX4 Pathway.

This study sought to explore potential roles of endothelial ferroptosis in radiation-associated atherosclerosis (RAA) and molecular mechanisms behind this phenomenon. Here, an in vivo RAA mouse model was used and treated with ferroptosis inhibitors. We found that the RAA group had a higher plaque burden and a reduction in endothelial cells with increased lipid peroxidation compared to the control group, while ameliorated by liproxstatin-1. In vitro experiments further confirmed that radiation induced the occurrence of ferroptosis in human artery endothelial cells (HAECs). Then, proteomics analysis of HAECs identified domain-containing protein 2 (DDHD2) as a co-differentially expressed protein, which was enriched in the lipid metabolism pathway. In addition, the level of lipid peroxidation was elevated in DDHD2-knockdown HAECs. Mechanistically, a significant decrease in the protein and mRNA expression of glutathione peroxidase 4 (GPX4) was observed in HAECs following DDHD2 knockdown. Co-immunoprecipitation assays indicated a potential interaction between DDHD2 and nuclear factor erythroid 2-related factor 2 (Nrf2). The downregulation of Nrf2 protein was also detected in DDHD2-knockdown HAECs. In conclusion, our findings suggest that radiation-induced endothelial ferroptosis accelerates atherosclerosis, and DDHD2 is a potential regulatory protein in radiation-induced endothelial ferroptosis through the Nrf2/GPX4 pathway.

Red blood cell-derived extracellular vesicles from type 2 diabetes patients induce endothelial dysfunction through a mechanism involving arginase 1

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Foundation for Geriatric Diseases Karolinska Institutet (2023-01860). Swedish Heart and Lung Foundation (20220210) Background The mechanisms driving the development of cardiovascular injury in type 2 diabetes (T2D) remain incompletely understood. We have recently demonstrated that red blood cells (RBCs) from patients with T2D (T2D-RBCs) act as mediators of endothelial dysfunction through the upregulation of arginase 1 and attenuation of nitric oxide bioavailability. However, the underlying mechanisms of this interaction remain unknown. It is increasingly clear that extracellular vesicles (EVs) are actively secreted by practically all cell types, including RBCs, and represent a novel mechanism of intercellular communication. However, the involvement of RBC-derived EVs in the development of endothelial dysfunction in T2D remains to be elucidated. Purpose To test the hypothesis that EVs are secreted by RBCs and transfer signalling to induce endothelial dysfunction in T2D through arginase 1. Methods EVs released from T2D-RBCs (T2D-RBCs EVs) and RBCs from age-matched healthy controls (H-RBCs EVs) were isolated using sequential ultracentrifugation or a membrane affinity column, co-incubated with mouse aortae to evaluate endothelium-dependent relaxation (EDR), and with human carotid artery endothelial cells (HCtAEC) to study the EV uptake and alteration in gene expression. EVs were characterized based on morphology, size and particle number, uptake in endothelial cells, and arginase 1 content. Functional involvement of EV uptake and arginase were investigated using pharmacological interventions. Arginase 1 was measured in EVs, HCtAEC, and mouse aortae after co-incubation with H-RBCs EVs and T2D-RBCs EVs. Results The uptake of T2D-RBCs EVs by endothelial cells was greater than that of EVs from H-RBCs (Fig. 1A, B) despite the reduced formation of EVs by T2D-RBCs (Fig. 1C). T2D-RBCs EVs significantly impaired EDR (Fig. 1D), and this impairment was prevented by inhibiting uptake of EVs with heparin (Fig. 1E). Arginase 1 was detected in RBC-derived EVs (Fig. 2A), and endothelial function was rescued by blocking arginase activity in EVs by the arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH; Fig. 2B). Immunohistochemical staining revealed upregulation of arginase 1 in the vasculature following incubation with T2D-RBCs EVs (Fig. 2C, D). Additionally, co-incubation of HCtAEC and EVs derived from T2D-RBCs significantly increased endothelial cell arginase 1 (Fig. 2E, F). Administration of ABH to the aortae following the co-incubation also attenuated the impairment of EDR induced by T2D-RBCs EVs, suggesting the involvement of vascular arginase 1 (Fig. 2G). Conclusion T2D-RBCs EVs induce endothelial dysfunction. In addition to increased uptake of EVs in endothelial cells, the signalling behind this effect of EVs is mediated by arginase 1 to induce endothelial dysfunction. These results shed new important light on the mechanism underlying vascular injury mediated by RBCs in T2D.

Abstract 2033: Bone Marrow-Derived Macrophage Extracellular Vesicles From Diabetic Mice Suppress Angiogenic Properties In Endothelial Cells

Introduction: Peripheral artery disease (PAD), increased in diabetes, leads to mortality and disability. Diabetes promotes a proinflammatory macrophage phenotype that suppresses repair. Macrophage-derived extracellular vesicles (EVs) have been associated with tissue damage. We hypothesize that diabetes perturbs macrophage EV cargo, thereby contributing to impaired repair responses to PAD/ischemia. Methods: Unilateral ligation of the left femoral artery (FAL) was performed to induce hind limb ischemia (HLI) in diabetic Akita (C57BL/6-Ins2 Akita /J) vs. non-diabetic (NDM, C57BL/6J) male mice. Murine BMDMs were grown in Exo-free medium. Ultracentrifugation was used to isolate EVs. Western blotting, transmission electron microscopy (TEM) and ZetaView nanoparticle tracking analysis were used to characterize EV size, number, concentration, and morphology. We profiled EV microRNA (miRNA) and protein cargos from Akita vs. NDM BMDM mice. Results: BMDMs-EV size was similar in Akita and NDM mice (diameter, 20-200nm). TEM showed cup-shaped morphology. Western blot confirmed known positive and negative control exosomal markers. Comparing Akita vs. NDM BMDM EVs, a total of 3277 proteins were identified; 821 proteins were significantly differentially expressed. Enrichment analysis highlighted pathways predominantly related to oxidative stress, insulin signaling, actin cytoskeleton, CLEAR, lipid metabolism, phagosome maturation, RNA binding and DNA replication. A total of 912 EV miRNAs were identified; 39 were significantly up-regulated and 19 were down-regulated. Preliminary analysis links a number of them to endothelial function and angiogenesis. In vitro , endothelial tube formation was impaired in human iliac artery endothelial cells treated with diabetic vs. NDM BMDM EVs. BMDM treated with diabetic vs. WT EVs displayed increased expression of inflammatory genes (TNF, IL-6,) and reduced expression of mitochondrial complex I and complex II proteins. Conclusion: We identified microRNAs and distinct proteins from diabetic EVs as potential mediators of impaired endothelial function and resolution of inflammation. Targeting macrophage EVs composition might identify therapeutic approaches to prevent and treat PAD in diabetes.

Abstract 1127: Erythrocyte Derived Extracellular Vesicles From Patients With Type 2 Diabetes Induce Endothelial Dysfunction Through Increased Vascular Oxidative Stress

Background: Red blood cells (RBCs) from patients with T2D (T2D-RBCs) induce endothelial dysfunction, but the mechanisms remain unclear. It is increasingly clear that extracellular vesicles (EVs) are actively secreted by RBCs and represent a novel mechanism of intercellular communication. However, the functional role of T2D-RBCs EVs in communication between RBCs and the cardiovascular system remains unknown. Purpose: This study aims to investigate whether EVs induce endothelial dysfunction in T2D and, if this is mediated via increased vascular oxidative stress. Material and Methods: EVs derived from T2D-RBCs and healthy RBCs (H-RBCs) were isolated using a membrane affinity column and co-incubated with wild-type mouse aortas for evaluation of the endothelium-dependent relaxation (EDR) using the wire myograph in the presence or absence of non-selective reactive oxygen species scavenger N-acetyl cysteine (NAC; 100 μM) applied to the aortas following the 18h EV incubation. The levels of the oxidative stress marker 4-hydroxynonenal (4-HNE) were quantified by immunohistochemistry in mouse aortas incubated with EVs. Additionally, T2D-RBCs EVs and H-RBCs EVs were co-incubated with human carotid artery endothelial cells (HCtAEC) for 8h and 24h to study the effects of EVs on vascular mRNA expression levels of eNOS, NOX1, and NOX4. Results: T2D-RBCs EVs but not H-RBCs EVs impaired EDR in aortas isolated from mice ( Fig. 1A ). This impairment was reversed by inhibiting oxidative stress in the vessel by NAC ( Fig. 1B ). Immunohistochemistry showed a significant increase in 4-HNE levels in aortas incubated with T2D-RBCs EVs ( Fig. 1C, D ). The T2D-RBCs EVs also induced a significant increase in NOX4 mRNA levels in HCtAEC after 24h but not 8h ( Fig. 1E, F ). However, T2D-RBCs EVs did not affect eNOS or NOX1 in endothelial cells. Conclusion: EVs derived from T2D-RBCs induce endothelial dysfunction through increased vascular oxidative stress.

Effects of Chronic Inflammatory Activation of Murine and Human Arterial Endothelial Cells at Normal Lipoprotein and Cholesterol Levels In Vivo and In Vitro.

The activation of endothelial cells is crucial for immune defense mechanisms but also plays a role in the development of atherosclerosis. We have previously shown that inflammatory stimulation of endothelial cells on top of elevated lipoprotein/cholesterol levels accelerates atherogenesis. The aim of the current study was to investigate how chronic endothelial inflammation changes the aortic transcriptome of mice at normal lipoprotein levels and to compare this to the inflammatory response of isolated endothelial cells in vitro. We applied a mouse model expressing constitutive active IκB kinase 2 (caIKK2)-the key activator of the inflammatory NF-κB pathway-specifically in arterial endothelial cells and analyzed transcriptomic changes in whole aortas, followed by pathway and network analyses. We found an upregulation of cell death and mitochondrial beta-oxidation pathways with a predicted increase in endothelial apoptosis and necrosis and a simultaneous reduction in protein synthesis genes. The highest upregulated gene was ACE2, the SARS-CoV-2 receptor, which is also an important regulator of blood pressure. Analysis of isolated human arterial and venous endothelial cells supported these findings and also revealed a reduction in DNA replication, as well as repair mechanisms, in line with the notion that chronic inflammation contributes to endothelial dysfunction.

Sulodeksyd w leczeniu retinopatii cukrzycowej

Diabetes mellitus is causing dysfunction of the vascular endothelial cells, which results in various pathologies within the microvascular and macrovascular system including retinopathy, neuropathy, nephropathy, and angiopathy. Endothelial dysfunction is caused by hyperglycemia via different mechanisms, such as damage to the glycocalyx, activation of the inflammation, oxidative stress, stimulation of cellular senescence, induction of imbalance between coagulation and fibrinolysis. Many studies suggests that inhibition of retinal endothelial dysfunction may be an effective way of slowing the progress of diabetic retinopathy. Sulodexide (glycosaminoglycans composed from heparin-like and dermatan fractions)is associated with a protective and regenerating action on the glycocalyx, slows down senescence of human venous and arterial endothelial cells. Moreover its mechanism of action involves an anti-inflammatory, anti-proteolytic, and antioxidant effect, including interference with metabolic and non-metabolic stress. In present study sulodexide was chosen to evaluate its therapeutic properties for the treatment of diabetic retinopathy.

Calciprotein Particles Induce Cellular Compartment-Specific Proteome Alterations in Human Arterial Endothelial Cells.

Calciprotein particles (CPPs) are indispensable scavengers of excessive Ca2+ and PO43- ions in blood, being internalised and recycled by liver and spleen macrophages, monocytes, and endothelial cells (ECs). Here, we performed a pathway enrichment analysis of cellular compartment-specific proteomes in primary human coronary artery ECs (HCAEC) and human internal thoracic artery ECs (HITAEC) treated with primary (amorphous) or secondary (crystalline) CPPs (CPP-P and CPPs, respectively). Exposure to CPP-P and CPP-S induced notable upregulation of: (1) cytokine- and chemokine-mediated signaling, Ca2+-dependent events, and apoptosis in cytosolic and nuclear proteomes; (2) H+ and Ca2+ transmembrane transport, generation of reactive oxygen species, mitochondrial outer membrane permeabilisation, and intrinsic apoptosis in the mitochondrial proteome; (3) oxidative, calcium, and endoplasmic reticulum (ER) stress, unfolded protein binding, and apoptosis in the ER proteome. In contrast, transcription, post-transcriptional regulation, translation, cell cycle, and cell-cell adhesion pathways were underrepresented in cytosol and nuclear compartments, whilst biosynthesis of amino acids, mitochondrial translation, fatty acid oxidation, pyruvate dehydrogenase activity, and energy generation were downregulated in the mitochondrial proteome of CPP-treated ECs. Differentially expressed organelle-specific pathways were coherent in HCAEC and HITAEC and between ECs treated with CPP-P or CPP-S. Proteomic analysis of mitochondrial and nuclear lysates from CPP-treated ECs confirmed bioinformatic filtration findings.

Abstract 18642: GWAS to Function Highlights the Role of REST in Endothelial Biology and Atherosclerosis

Introduction: Despite the emergence of new lipid-lowering therapies, coronary artery disease (CAD) remains the leading cause of death worldwide. Genome-wide association studies (GWAS) for CAD have discovered multiple novel loci that contribute to CAD pathogenesis. However, limitations in the interpretability of those GWAS associations have hindered the clinical translation of those findings. Hypothesis: In this study, we sought to evaluate the mechanisms of the association between chromosomal locus 4q12 and CAD, replicated in multiple independent GWAS for CAD. Methods: We utilized a combination of computational methods and in vitro assays to achieve our objectives. Results: Meta-analysis of public expression quantitative trait locus (eQTL) data and multi-trait colocalization across atherosclerosis-relevant cell types revealed that the CAD 4q12 locus regulates the expression of REST, a transcriptional repressor, in human aortic endothelial cells (HAEC) and prioritized 2 enhancer variants as candidate causal variants for the GWAS association. CRISPR interference confirmed that the rs6853156 variant regulates REST expression in inducible human endothelial cells. Pathway enrichment analysis in HAEC after REST silencing showed that epithelial to mesenchymal transition was the most upregulated pathway suggesting a potential mechanism via which silencing REST contributes to atherosclerosis. Endothelial to mesenchymal transition (endMT) was confirmed in HAEC after REST silencing at the RNA and protein level by rt-qPCR, western blot, and immunofluorescence. Concordantly, REST silencing increased endothelial permeability and matrix metalloproteinase activity in endothelial cell supernatants in vitro. L1CAM, a known endMT activator was only expressed in HAEC upon REST silencing. Chromatin immunoprecipitation sequencing supports direct binding of REST to the L1CAM promoter. Simultaneous silencing of L1CAM and REST in HAEC inhibited the observed upregulation of mesenchymal genes induced by REST silencing. Conclusions: In summary, our data reveal a novel role of REST as a repressor that functions as a constitutive inhibitor of endMT in human arterial endothelial cells.

Multi-Omics Profiling of Human Endothelial Cells from the Coronary Artery and Internal Thoracic Artery Reveals Molecular but Not Functional Heterogeneity.

Major adverse cardiovascular events occurring upon coronary artery bypass graft surgery are typically accompanied by endothelial dysfunction. Total arterial revascularisation, which employs both left and right internal thoracic arteries instead of the saphenous vein to create a bypass, is associated with better mid- and long-term outcomes. We suggested that molecular profiles of human coronary artery endothelial cells (HCAECs) and human internal mammary artery endothelial cells (HITAECs) are coherent in terms of transcriptomic and proteomic signatures, which were then investigated by RNA sequencing and ultra-high performance liquid chromatography-mass spectrometry, respectively. Both HCAECs and HITAECs overexpressed molecules responsible for the synthesis of extracellular matrix (ECM) components, basement membrane assembly, cell-ECM adhesion, organisation of intercellular junctions, and secretion of extracellular vesicles. HCAECs were characterised by higher enrichment with molecular signatures of basement membrane construction, collagen biosynthesis and folding, and formation of intercellular junctions, whilst HITAECs were notable for augmented pro-inflammatory signaling, intensive synthesis of proteins and nitrogen compounds, and enhanced ribosome biogenesis. Despite HCAECs and HITAECs showing a certain degree of molecular heterogeneity, no specific markers at the protein level have been identified. Coherence of differentially expressed molecular categories in HCAECs and HITAECs suggests synergistic interactions between these ECs in a bypass surgery scenario.

Thrombin receptor PAR1 silencing in endothelial colony–forming cells modifies stemness and vasculogenic properties

BackgroundThe involvement of thrombin receptor PAR1 in blood vessel development has been largely demonstrated in knockout mice; however, its implication in adult mouse angiogenesis seems very moderate. ObjectivesWe aimed to explore the potential relationships between PAR1, stemness, and angiogenic properties of human endothelial colony–forming cells (ECFCs). Methods and ResultsPAR1 activation on ECFCs using the selective PAR1-activating peptide induced a significant decrease in CD133 expression (RTQ-PCR analysis). In line, silencing of PAR1 gene expression with siRNA increased CD133 mRNA as well as intracellular CD133 protein expression. To confirm the link between CD133 and PAR1, we explored the association between PAR1 and CD133 levels in fast and slow fibroblasts prone to reprogramming. An imbalance between PAR1 and CD133 levels was evidenced, with a decreased expression of PAR1 in fast reprogramming fibroblasts expressing a high CD133 level. Regarding in vitro ECFC angiogenic properties, PAR1 silencing with specific siRNA induced cell proliferation evidenced by the overexpression of Ki67. However, it did not impact migration properties nor ECFC adhesion on smooth muscle cells or human arterial endothelial cells. In a mouse model of hind-limb ischemia, PAR1 silencing in ECFCs significantly increased postischemic revascularization compared to siCtrl-ECFCs along with a significant increase in cutaneous blood flows (P < .0001), microvessel density (P = .02), myofiber regeneration (P < .0001), and human endothelial cell incorporation in muscle (P < .0001). ConclusionIn conclusion, our work describes for the first time a link between PAR1, stemness, and vasculogenesis in human ECFCs.

Role of simulated in vitro gastrointestinal digestion on biotransformation and bioactivity of astragalosides from Radix Astragali

Radix Astragali (RA) is commonly used in Asian herbal therapy or food supply, and astragalosides and flavonoids are its major components with diverse pharmaceutical effects. To provide new information on the potential cardiovascular benefits of RA administered orally, the bioaccessibility of these compounds with relevant in vitro digestion parameters was determined for four digestion phases (oral, gastric, small and large intestines) by ultrahigh-performance liquid chromatography quadrupole time-of-flight-mass spectrometry (UPLC-Q-TOF/MS). Meanwhile, we compared the effects of digestion products on advanced glycation end products (AGEs)-induced intracellular reactive oxygen species (ROS) levels in a human arterial endothelial cells (HAECs) model, and studied the potential of RA against oxidative stress-related cardiovascular disease. The changes of saponins and flavonoids composition and antioxidant activity after digestion in intestines were mainly due to the astragaloside IV (AS-IV) biosynthesis involving saponins acetyl isomerization and deacetylation, and the flavonoid glycosides converted to aglycone by deglycosylation processes. All these results suggest that acetyl biotransformation of RA in small intestine directly influenced the response to oxidative stress, and might provide a reference for elucidation of the multi-component action after oral RA in cardiovascular health care.

Whole-Genome Transcriptome Profiling in PFOS-treated Uterine Artery Endothelial Cells Isolated from Pregnant Women.

Empirical evidence from human studies has demonstrated a correlative relationship between perfluorooctane sulfonate (PFOS) exposure and increased risks of preeclampsia and fetal developmental complications. Although experimental and circumstantial data suggest that PFOS induces endothelial dysfunction, leading to decreased uterine arterial blood flow and gestational hypertension, the precise regulatory mechanisms responsible for this effect remain unknown. To address this issue, we treated human uterine artery endothelial cells (hUAECs) isolated from pregnant women with 10 μmol/L PFOS or vehicle and conducted comparative transcriptomic analyses. We identified a total of 19 differentially expressed genes, 9 of which were upregulated and 10 were down-regulated in PFOS-treated pregnant hUAECs. Pre-ranked gene set enrichment analysis unveiled a distinct set of activated genes involved in osmotic stress, cellular stress response, translation regulation, metabolic regulation, and oxidation-reduction processes in PFOS-treated pregnant hUAECs. Furthermore, PFOS treatment resulted in the downregulation of genes implicated in cardiac muscle cell proliferation, embryonic morphogenesis, and muscle cell proliferation. In addition, we observed differential splicing events in 2678 genes in hUAECs exposed to PFOS, with cross-comparison analysis revealing 4 genes that were both differentially expressed and alternatively spliced and were implicated in oxidative stress and cardiac development. In conclusion, this study provides a comprehensive understanding of the molecular mechanisms underlying PFOS-induced gestational uterine artery endothelial dysfunction during pregnancy, offering a valuable resource for future research in this field.

Abstract 13269: Effects of Nicotine-Containing Product Challenges on Interleukin-6 Pathway Expression in Human Pluripotent-Derived Arterial Endothelial Cells: The Clues Study

Background: Electronic nicotine delivery devices (ENDs) induce interleukin-6 (IL-6) pathway expression in airway epithelial cells, but their systemic effects on arterial endothelial cells (AECs) are unknown. We hypothesized differential expression (DE) of IL-6 pathway genes by human pluripotent-derived AECs exposed in vitro to participant serum drawn before and after use of ENDS by chronic vapers, use of combustible cigarettes by smokers, and no product use by controls. Methods: For this pilot study, we randomly selected 30 participants from the CLUES Study: 10 exclusive vapers (exhaled carbon monoxide [CO] &lt;5 ppm, + urine NicCheck I), 10 exclusive smokers (CO &gt;5 ppm, + urine), and 10 non-vaping/non-smoking controls (CO &lt;5 ppm, negative urine). We generated purified populations of AECs (H9 CPTC C13 derivative human embryonic stem cell line) and exposed them for 6 hours to 50% concentrated participant serum drawn before and 15’ after a product use challenge. Total RNA was sequenced on a NextSeq 2000. The DESeq2 suite characterized DE with an a priori focus on genes in the IL-6 biosynthetic pathway gene ontology set. Z scores of normalized log2 gene expression values were determined and expressed as heat maps (Figure). Gene Set Variational Analysis (GSVA) was used to interrogate for enriched gene sets. P values were not adjusted for multiple hypothesis testing and are exploratory. Results: Vapers were a mean (SD) 28.9 (8.1) years old (30% female), smokers were 38.3 (11.6) years old (40% female), and controls were 33.6 (17.7) years old (50% female). Compared to controls, vapers had increased expression of the IL-6 pathway at baseline (p=0.004), but smokers did not (p=0.38). Following product use, vapers (p=0.0009) but not smokers (p=0.79) had increased expression of genes in the IL-6 biosynthetic pathway (Figure). Conclusion: Chronic vapers, but not smokers, may have increased expression of genes in the IL-6 pathway prior to product use with further increases afterwards.

Circulating CD31 reflects endothelial activity and is associated with a lower risk for cardiovascular complications

Abstract Background CD31 is a membrane receptor expressed by leucocytes, platelets and endothelial cells. CD31 regulates endothelial leukocyte transmigration and is key in maintaining endothelial homeostasis. An increased endothelial activation is suggested to contribute to atherosclerotic plaque inflammation but studies focusing on CD31 in atherosclerosis have provided evidence for both pro- and anti-atherosclerotic effects. However, which biological processes that are reflected by circulating soluble CD31 (sCD31) in human atherosclerosis remain unknown. Purpose Here, we aimed to explore 1) which biological processes circulating sCD31 reflects in the human atherosclerotic plaque and circulation 2) if sCD31 levels predicts cardiovascular death in individuals with carotid atherosclerosis and 3) if circulating sCD31 is causally associated to coronary artery disease (CAD) and ischemic stroke, in order to investigate its role as a biomarker of atherosclerotic disease and complications. Methods Cardiovascular associated plasma proteins (n=543) and carotid plaque proteins (n=197) were assessed by a proximity extension assay (Olink, SciLife) in the Carotid Plaque Imaging Project biobank. Plaques were phenotyped by immunohistochemistry. CD31 was located by immunofluorescence. Human carotid artery endothelial cells (HCtAECs) were stimulated with IL-6 and TNF-α to assess sCD31 release. National registries were used to identify cardiovascular death during follow up. All patients gave written informed consent. The study was approved by the local ethical review board and followed the declaration of Helsinki. Causal effects for sCD31 on stroke and CAD were examined by Mendelian randomization (MR). Summary statistics for GWAS of circulating CD31, coronary artery disease and stroke were retrieved from the SCALLOP study, the CARDIoGRAMplusC4D and the MEGASTROKE consortia. Results Plasma sCD31 correlated to plaque sCD31, TNF-α, MMP-2, MMP-9 levels and CD68+ plaque area. Plasma sCD31 also correlated to plasma CSF-1, MCP-1 and IL-8 levels. Clustering and enrichment analyses revealed that plasma sCD31 was associated to angiogenesis, cell migration and inflammatory responses. CD31 was pre-dominantly expressed on the plaque endothelium. TNF-α and IL-6 induced HCtAECs sCD31 release in vitro. Furthermore, low levels of circulating sCD31 predicted cardiovascular death and our MR analyses suggested that low circulating sCD31 levels were causally associated with a higher risk of stroke and CAD. Conclusions The present study shows that circulating sCD31 is released from endothelial cells due to inflammatory stimuli and reflects plaque inflammation. However, our follow-up data and MR analyses suggest that circulating sCD31 is associated with a lower risk for cardiovascular complications. This indicates that circulating sCD31 is not only a marker of an inflammatory response but also a marker of a functional endothelium, able to maintain the endothelial homeostasis. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): The Swedish Society for Medical Research, The Swedish Research Council, Crafoord foundation, The Swedish Society of Medicine, The Swedish Heart and Lung Foundation, The Knut and Alice Wallenberg foundation, the Medical Faculty at Lund University and Region Skåne.

Carotid endarterectomy plaques of patients with elevated levels of lipoprotein(a) demonstrate increased intraplaque angiogenesis

Background and Aims : Elevated lipoprotein(a) [Lp(a)] levels have been demonstrated to be a causal risk factor for the development of cardiovascular disease. Previously, we described that patients with elevated Lp(a) levels have an increased risk of major adverse cardiovascular events (MACE), mostly stroke, 30 days after undergoing carotid endarterectomy (CEA). Aim: Unravelling the mechanism underlying this increase in MACE.Methods: We performed targeted plasma proteomics analysis using the proximity extension assay for 276 proteins on complete plasma from patients undergoing CEA with high levels (>195 mg/dl; N=39) and low levels (<7 mg/dl; N=73) of Lp(a).Results: Surprisingly, no differences were observed in the expression of systemic plasma proteins. Hence, we started to assess the local effects of Lp(a) on plaque morphology. Plaque phenotyping demonstrated that patients with high levels (>195 mg/dl; N=57) of Lp(a) had a 33% increase in mean vessel density compared to patients with low levels (<7 mg/dl; N=106), as attested by semi-quantitatively scoring of the plaques. This increase in neovascularization correlates with decreased plaque stability, as was indicated by increased intraplaque haemorrhage. In-vitro stimulation of Human Arterial Endothelial Cells with physiologically relevant levels of Lp(a) (100 mg/dl), results in enhanced collagen degradation capacity, hyper-sprouting in a 3D spheroid assay and increased blood vessel invasion using a 3D collagen-invasiveness assay. Currently, we are elucidating the signalling pathways underlying this Lp(a)-induced angiogenesis, which we strive to present at the EAS 2022.Conclusions: Lp(a)-induced intraplaque angiogenesis can contribute to the increased risk of major adverse cardiovascular events in patients with elevated levels of Lp(a). Background and Aims : Elevated lipoprotein(a) [Lp(a)] levels have been demonstrated to be a causal risk factor for the development of cardiovascular disease. Previously, we described that patients with elevated Lp(a) levels have an increased risk of major adverse cardiovascular events (MACE), mostly stroke, 30 days after undergoing carotid endarterectomy (CEA). Aim: Unravelling the mechanism underlying this increase in MACE. Methods: We performed targeted plasma proteomics analysis using the proximity extension assay for 276 proteins on complete plasma from patients undergoing CEA with high levels (>195 mg/dl; N=39) and low levels (<7 mg/dl; N=73) of Lp(a). Results: Surprisingly, no differences were observed in the expression of systemic plasma proteins. Hence, we started to assess the local effects of Lp(a) on plaque morphology. Plaque phenotyping demonstrated that patients with high levels (>195 mg/dl; N=57) of Lp(a) had a 33% increase in mean vessel density compared to patients with low levels (<7 mg/dl; N=106), as attested by semi-quantitatively scoring of the plaques. This increase in neovascularization correlates with decreased plaque stability, as was indicated by increased intraplaque haemorrhage. In-vitro stimulation of Human Arterial Endothelial Cells with physiologically relevant levels of Lp(a) (100 mg/dl), results in enhanced collagen degradation capacity, hyper-sprouting in a 3D spheroid assay and increased blood vessel invasion using a 3D collagen-invasiveness assay. Currently, we are elucidating the signalling pathways underlying this Lp(a)-induced angiogenesis, which we strive to present at the EAS 2022. Conclusions: Lp(a)-induced intraplaque angiogenesis can contribute to the increased risk of major adverse cardiovascular events in patients with elevated levels of Lp(a).

Neutrophil Elastase Increases Vascular Permeability and Leukocyte Transmigration in Cultured Endothelial Cells and Obese Mice.

Neutrophil elastase (NE) plays a pivotal role in inflammation. However, the mechanism underlying NE-mediated inflammation in obesity remains unclear. Here, we report that NE activates protease-activated receptor-2 (PAR2), stimulates actin filament (F-actin) formation, decreases intercellular junction molecule VE-cadherin expression, and increases the permeability of human arterial endothelial cells (hECs). NE also prompts degradation of VE-cadherin and its binding proteins p120- and β-catenins via MG132-sensitive proteasomes. NE stimulates phosphorylation of myosin light-chain (MLC) and its regulator myosin phosphatase target subunit-1 (MYPT1), a target of Rho kinase (ROCK). Inhibitors of PAR2 and ROCK prohibit NE-induced F-actin formation, MLC phosphorylation, and VE-cadherin reduction in hECs, and impede monocyte transmigration through hEC monolayer pretreated with either neutrophils or NE. Further, administration of an NE inhibitor GW311616A significantly attenuates vascular leakage, leukocyte infiltration, and the expression of proinflammatory cytokines in the white adipose tissue from high-fat diet (HFD)-induced obese mice. Likewise, NE-deficient mice are resistant to HFD-induced vascular leakage in the heart. Together, NE regulates actomyosin cytoskeleton activity and VE-cadherin expression by activating PAR2 signaling in the endothelial cells, leading to increased vascular permeability and leukocyte extravasation. Hence, inhibition of NE is a potential approach to mitigate vascular injury and leukocyte infiltration in obesity-related systemic inflammation.

Generating human artery and vein cells from pluripotent stem cells highlights the arterial tropism of Nipah and Hendra viruses

Stem cell research endeavors to generate specific subtypes of classically defined "cell types." Here, we generate >90% pure human artery or vein endothelial cells from pluripotent stem cells within 3-4days. We specified artery cells by inhibiting vein-specifying signals and vice versa. These cells modeled viral infection of human vasculature by Nipah and Hendra viruses, which are extraordinarily deadly (∼57%-59% fatality rate) and require biosafety-level-4 containment. Generating pure populations of artery and vein cells highlighted that Nipah and Hendra viruses preferentially infected arteries; arteries expressed higher levels of their viral-entry receptor. Virally infected artery cells fused into syncytia containing up to 23 nuclei, which rapidly died. Despite infecting arteries and occupying ∼6%-17% of their transcriptome, Nipah and Hendra largely eluded innate immune detection, minimally eliciting interferon signaling. We thus efficiently generate artery and vein cells, introduce stem-cell-based toolkits for biosafety-level-4 virology, and explore the arterial tropism and cellular effects of Nipah and Hendra viruses.

The Distinct Role of the HDL Receptor SR-BI in Cholesterol Homeostasis of Human Placental Arterial and Venous Endothelial Cells.

As opposed to adults, high-density lipoprotein (HDL) is the main cholesterol carrying lipoprotein in fetal circulation. The major HDL receptor, scavenger receptor class B type I (SR-BI), contributes to local cholesterol homeostasis. Arterial endothelial cells (ECA) from human placenta are enriched with cholesterol compared to venous endothelial cells (ECV). Moreover, umbilical venous and arterial plasma cholesterol levels differ markedly. We tested the hypothesis that the uptake of HDL-cholesteryl esters differs between ECA and ECV because of the differential expression of SR-BI. We aimed to identify the key regulators underlying these differences and the functional consequences. Immunohistochemistry was used for visualization of SR-BI in situ. ECA and ECV were isolated from the chorionic plate of human placenta and used for RT-qPCR, Western Blot, and HDL uptake assays with 3H- and 125I-labeled HDL. DNA was extracted for the methylation profiling of the SR-BI promoter. SR-BI regulation was studied by exposing ECA and ECV to differential oxygen concentrations or shear stress. Our results show elevated SR-BI expression and protein abundance in ECA compared to ECV in situ and in vitro. Immunohistochemistry demonstrated that SR-BI is mainly expressed on the apical side of placental endothelial cells in situ, allowing interaction with mature HDL circulating in the fetal blood. This was functionally linked to a higher increase of selective cholesterol ester uptake from fetal HDL in ECA than in ECV, and resulted in increased cholesterol availability in ECA. SR-BI expression on ECV tended to decrease with shear stress, which, together with heterogeneous immunostaining, suggests that SR-BI expression is locally regulated in the placental vasculature. In addition, hypomethylation of several CpG sites within the SR-BI promoter region might contribute to differential expression of SR-BI between chorionic arteries and veins. Therefore, SR-BI contributes to a local cholesterol homeostasis in ECA and ECV of the human feto-placental vasculature.

AT2R activation increases in vitro angiogenesis in pregnant human uterine artery endothelial cells.

Angiogenesis is vital during pregnancy for remodeling and enhancing vasodilation of maternal uterine arteries, and increasing uterine blood flow. Abnormal angiogenesis is associated with decreased uteroplacental blood flow and development of pregnancy disorders such as gestational hypertension, preeclampsia, fetal growth restriction, preterm delivery, stillbirth, and miscarriage. The mechanisms that contribute to normal angiogenesis remain obscure. Our previous studies demonstrated that expression of the angiotensin type 2 receptor (AT2R) is increased while the angiotensin type 1 receptor (AT1R) is unchanged in the endothelium of uterine arteries, and that AT2R-mediated pregnancy adaptation facilitates enhanced vasodilation and uterine arterial blood flow. However, the role of AT2R in regulating angiogenesis during pregnancy has never been studied. This study examines whether or not AT2R activation induces angiogenesis and, if so, what mechanisms are involved. To this end, we used primary human uterine artery endothelial cells (hUAECs) isolated from pregnant and nonpregnant women undergoing hysterectomy. The present study shows that Compound 21, a selective AT2R agonist, induced proliferation of pregnant-hUAECs, but not nonpregnant-hUAECs, in a concentration-dependent manner, and that this C21-induced mitogenic effect was blocked by PD123319, a selective AT2R antagonist. The mitogenic effects induced by C21 were inhibited by blocking JNK—but not ERK, PI3K, and p38—signaling pathways. In addition, C21 concentration dependently increased cell migration and capillary-like tube formation in pregnant-hUAECs. The membrane-based antibody array showed that C21 increased expression of multiple angiogenic proteins, including EGF, bFGF, leptin, PLGF, IGF-1, and angiopoietins. Our qPCR analysis demonstrates that C21-induced increase in expression of these angiogenic proteins correlates with a proportional increase in mRNA expression, indicating that AT2R activates angiogenic proteins at the transcriptional level. In summary, the present study shows that AT2R activation induces angiogenesis of hUAECs in a pregnancy-specific manner through JNK-mediated pathways with associated transcriptional upregulation of multiple proangiogenic proteins.