Carotid Artery Endothelial Cells Research Articles

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 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.

Development of a novel Guinea Pig model producing transgenerational endothelial transcriptional changes driven by maternal food restriction and a second metabolic insult of high fat diet

Developmental programming of chronic adverse cardiovascular health outcomes has been studied both using numerous human populations and an array of animal models. However, the mechanisms that produce transgenerational effects have been difficult to study due to a lack of developmentally relevant models. As such, how increased disease risk is carried to the second generation has been poorly studied. We hypothesized that the endothelium which mediates many acute and chronic vascular inflammatory responses is a key player in these effects, and epidemiological studies implicate transgenerational nutritional effects on endothelial health. To study the mutigenerational effects of maternal undernutrition on offspring endothelial health, we developed a model of transgenerational nutritional stress in guinea pigs, a translationally relevant precocial species with a relatively short lifespan. First- and second-generation offspring were subjected to a high fat diet in adolescence to exacerbate negative cardiovascular health. To assess transcriptional changes, we performed bulk RNA-sequencing in carotid artery endothelial cells, with groups stratified as prenatal control or food restricted, and postnatal control or high fat diet. We detected statistically significant gene alterations for each dietary permutation, some of which were unique to treatments and other transcriptional signatures shared by multiple or all conditions. These findings highlight a core group of genes altered by high fat diet that is shared by all cohorts and a divergence of transgenerational effects between the prenatal ad libitum and dietary restriction groups. This study establishes the groundwork for this model to be used to better understand the interplay of prenatal stress and genetic reprogramming.

INFLUENCE OF OBESITY ON VASCULAR DYSFUNCTION AFTER TRAUMATIC HEMORRHAGE.

Background: Obesity increases the risk for morbidity and mortality after trauma. These complications are associated with profound vascular damage. Traumatic hemorrhage acutely attenuates vascular responsiveness, but the impact of obesity on this dysfunction is not known. The local inflammatory response in vascular cells is also unknown. We hypothesized that obesity potentiates trauma-induced vascular inflammation and dysfunction. Methods: Male Sprague-Dawley rats (~250 g) were fed normal chow (NC; 13.5% kcal fat, n = 20) or high-fat (HF; 60% kcal fat, n = 20) diets for 6 to 8 weeks. Under anesthesia, hemorrhage was induced by a mesenteric artery laceration, a Grade V splenic injury, and hypotension (MAP = 30-40 mm Hg) for 30 minutes. Vascular responsiveness was assessed ex vivo in isolated mesenteric arteries prehemorrhage and posthemorrhage. Gene expression for IL-1β, and IL-6, prooxidant nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2), and α-adrenergic receptor were assessed in carotid artery endothelial cells (ECs) and non-ECs (media + adventitia). Results: In NC rats, hemorrhage attenuated norepinephrine-induced vasoconstriction and endothelium-dependent vasodilation to acetylcholine. In HF rats, baseline norepinephrine-induced vasoconstriction was attenuated compared with NC, but vasoconstriction and endothelium-dependent vasodilation did not change prehemorrhage to posthemorrhage. Hemorrhage led to elevated IL-1β gene expression in ECs and elevated IL1β, IL-6, NOX2, and α-adrenergic receptor gene expression in the media + adventitia compared with sham. HF rats had greater EC IL-1 β and NOX2 gene expression compared with NC rats. The hemorrhage-induced elevation of IL-1β in the media + adventitia was greatest in HF rats. Conclusion: Traumatic hemorrhage attenuates vascular responsiveness and induces vascular inflammation. The attenuated vascular responsiveness after hemorrhage is absent in obese rats, while the elevated vascular inflammation persists. A HF diet amplifies the arterial inflammation after hemorrhage. Altered vascular responsiveness and vascular inflammation may contribute to worse outcomes in obese trauma patients.

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.

Exacerbated Vascular Inflammation Following Traumatic Hemorrhage in Obese Rats

IntroductionObesity increases the risk for morbidity and mortality in trauma patients. These complications are associated with prolonged tissue hypoperfusion, hypercoagulability, and edema that reflect profound vascular damage. Traumatic hemorrhage acutely attenuates vascular responsiveness, but the degree of this dysfunction during the obese state is not known. Traumatic hemorrhage also increases circulating inflammatory cytokines; however, the local inflammatory response in vascular cells is unknown. We hypothesized that obesity potentiates trauma‐induced vascular inflammation and dysfunction.MethodsMale Sprague‐Dawley rats (~250g) fed either normal chow (NC; 13.5% kcal fat, n=20), moderate‐fat (MF; 33% kcal fat, n=20), or high‐fat (HF; 60% kcal fat, n=20) diet for 6‐8 weeks were assigned to a traumatic hemorrhage protocol or sham control. Under anesthesia, hemorrhage was induced by the laceration of a mesenteric artery and a Grade V splenic injury. Hypotension (mean arterial pressure = 30–40 mm Hg) was maintained for 30 minutes. Vasoconstrictor and vasodilator properties were assessed ex vivo in isolated mesenteric arteries pre‐ and post‐ hemorrhage. Carotid arteries collected after hemorrhage or sham control were flushed with trizol to collect endothelial cells (ECs). Gene expression was assessed in ECs and the remaining carotid artery (media + adventitia) cells for pro‐inflammatory interleukin (IL)‐1β, and IL‐6, pro‐oxidant NADPH oxidase 2 (NOX2), and α‐adrenergic receptor.ResultsIn NC rats, hemorrhage attenuated norepinephrine‐induced vasoconstriction and endothelium‐dependent dilation. This was accompanied by elevated IL‐1β expression in carotid artery ECs and elevated IL‐1β, IL‐6, NOX2, and α‐adrenergic receptor expression in the carotid artery media + adventitia post‐hemorrhage versus sham control. In MF and HF rats, norepinephrine‐induced vasoconstriction and endothelium‐dependent dilation did not change pre‐ to post‐hemorrhage. In sham animals, endothelial cell IL‐1β and NOX2 expression were higher in HF compared with NC rats. Similar to NC rats, the MF and HF rats had higher IL‐1β expression in carotid artery ECs, and IL‐1β, IL‐6, NOX2, and α‐adrenergic receptor expression in the carotid artery media + adventitia post‐hemorrhage compared with sham. The hemorrhage‐induced elevation of IL‐1β in carotid artery ECs and media + adventitia was greater in HF rats compared with NC and MF rats.ConclusionOur clinically‐relevant model of traumatic hemorrhage attenuates vascular responsiveness and induces vascular inflammation. The attenuated vascular responsiveness following hemorrhage is absent in MF and HF rats, while the elevated vascular inflammation persists. HF rats have an amplified arterial inflammatory response to hemorrhage, both in ECs and in cells of the media + adventitia. The augmented vascular inflammatory response to hemorrhage in HF rats may explain the worse outcomes in obese trauma patients.

Hsa_circ_0003204 Knockdown Weakens Ox-LDL-Induced Cell Injury by Regulating miR-188-3p/TRPC6 Axis in Human Carotid Artery Endothelial Cells and THP-1 Cells.

Background: Circular RNAs (circRNAs) are involved in atherosclerosis (AS) development. However, the function and mechanism of circRNA hsa_circ_0003204 (circ_0003204) in carotid artery AS remain unclear.Methods: Oxidized low-density lipoprotein (ox-LDL)-treated human carotid artery endothelial cells (HCtAECs) and THP-1 cells were used as cell models of carotid artery AS. Relative levels of circ_0003204, microRNA-188-3p (miR-188-3p), and transient receptor potential canonical channel 6 (TRPC6) were detected by quantitative reverse transcription–polymerase chain reaction or Western blotting. The targeting relationship between circ_0003204 or TRPC6 and miR-188-3p was assessed via dual-luciferase reporter analysis and RNA immunoprecipitation. Cell proliferation was assessed via 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay and 5-ethynyl-2′-deoxyuridine (EdU) assay. Cell apoptosis was analyzed via assessing cell caspase-3 activity, apoptosis, and apoptosis-related protein. Inflammatory response was analyzed via analysis of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α). Oxidative stress was assessed via determination of reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD).Results: Circ_0003204 and TRPC6 levels were elevated, and miR-188-3p expression declined in ox-LDL-treated HCtAECs and THP-1 cells. Circ_0003204 could regulate TRPC6 expression via mediating miR-188-3p. Circ_0003204 silencing weakened ox-LDL-induced viability inhibition and apoptosis in HCtAECs, and inflammatory response and oxidative stress in THP-1 cells via regulating miR-188-3p. MiR-188-3p overexpression attenuated ox-LDL-induced injury in HCtAECs and THP-1 cells by targeting TRPC6.Conclusion: Circ_0003204 knockdown mitigated ox-LDL-induced injury in HCtAECs and THP-1 cells via regulating the miR-188-3p/TRPC6 axis, indicating that circ_0003204 might play an important role in carotid artery AS.

Effects of in‐vitro exercise‐induced endothelial shear stress on adhesion molecule gene expression

BackgroundExercise‐Induced Shear Stress (ESS) is characterized as a mechanical stimulus effecting cellular morphology and physiology. Varying ESS intensity elicit different response mechanisms through cellular signaling pathways. Higher intensity ESS enhances the expression of genes involved in protection against atherosclerosis. Adhesion molecules expression is common in atherosclerosis. Therefore, the purpose of this study was to determine the effects of different intensities of pulsatile ESS on vascular cell adhesion molecule‐1 (VCAM1) and intercellular adhesion molecule (ICAM1) gene expression.MethodsCommercially available human carotid artery endothelial cells (Sigma Aldrich) were cultured until 95–100% confluence and then exposed to pulsatile resting ESS (18 dynes/cm2) for 5 hours, followed by 1 hour low‐intensity ESS (35 dynes/cm2), or high‐intensity ESS (80 dynes/cm2) on parallel fluidic units (Ibidi, USA). Ibidi Pump Control Software (version 1.5.4, Ibidi, USA) was used to control shear stress intensity. mRNA was extracted and expression of VCAM1 and ICAM1 was assessed using qRT‐PCR. Samples were run in Changes in gene expression were calculated using the ΔΔ‐CT method, using GAPDH as the normalizing control gene. One‐way ANOVA was used to asses statistically significant differences (p < 0.05) using Prism version 8 (Graphpad).ResultsThe mRNA expression of VCAM1 and ICAM1 significantly increased (p <0.01). 6 and 8 folds respectively after exposure to low‐intensity ESS. Furthermore, high‐intensity ESS did not significantly increased the expression of VCAM1 and ICAM1.ConclusionLow‐intensity pulsatile exercise‐induced shear stress improves endothelial function in‐vitro through the increase in adhesion molecule’s gene expression.

Effects of in Vitro Exercise‐Induced Endothelial Shear Stress on Oxidative Stress and Vasoconstriction Gene Expression

BackgroundSedentary lifestyle contributes to endothelial dysfunction through increasing oxidative stress and endothelin‐1, a potent vasoconstrictor that contributes to the pathogenesis of vascular disease. Exercise‐induced endothelial shear stress (ESS) elicits a mechanical stimulus leading to intracellular signaling transduction regulating cellular function that might protect against endothelial dysfunction through promotion of the expression of anti‐oxidative and pro‐vasodilatory genes. Although it has been reported that increments on steady ESS has led to improvements in endothelial function in vivo, the role of pulsatile exercise induced ESS and varying exercise intensities in vitro has not been explored yet. Therefore, the purpose of this study was to determine the effects of pulsatile exercise‐induced ESS based on in vivo exercise intensity on anti‐oxidative (superoxide dismutase, SOD2) and vasoconstrictive (endothelin 1, EDN‐1) gene expression.MethodsCommercially available human carotid artery endothelial cells (Sigma‐Aldrich Comp.) were cultured until 95–100% confluence and exposed to pulsatile resting ESS (18 dynes/cm2) for 5 hours, followed by 1 hour at resting ESS, low‐intensity exercise‐induced ESS (35 dynes/cm2), or high‐intensity exercise‐induced ESS (80 dynes/cm2) on parallel fluidic units (Ibidi USA, Inc.). Ibidi pump software (PumpControl Software 1.5.4) was used to control shear stress intensity. Total RNA was extracted and reverse transcribed followed by measurement of EDN‐1 and SOD2 mRNA expression via qRT‐PCR. qRT‐PCRs were performed in duplicates and changes in gene expression were calculated using the ΔΔ‐CT method with GAPDH used as the normalizing control gene. Statistical analysis was performed using graphpad prism 8 software (Graphpad Software.). Significance was considered at p<0.05.ResultsmRNA expression of SOD2 increased 2.5‐fold after exposure to low‐intensity exercise‐induced ESS (p<0.05). EDN‐1 showed a 5‐fold increase following exposure to low‐intensity exercise‐induced ESS (p<0.05) in comparison to resting conditions. High‐intensity pulsatile exercise‐induced ESS decreased the expression of EDN‐1 in comparison to resting and low‐intensity conditions (p<0.05).ConclusionPulsatile exercise‐induced ESS improved anti‐oxidative properties in human carotid artery endothelial cells. It seems that higher exercise intensities are required to downregulate the expression of vasoconstrictive genes.

P51 Effects of Pulsatile Exercise-induced Shear Stress on eNOS, SOD, VCAM-1, and ICAM-1 mRNA Expression of Human Carotid Artery Endothelial Cells

Exercise-induced endothelial shear stress (ESS) has been proposed as a molecular mechanism that regulates the expression of genes involved in the protection against atherosclerosis. However, research on this topic has not considered the pulsatile nature of blood flow for in vivo ESS estimations.PurposeTo analyze the effect of pulsatile exercise-induced ESS on endothelial nitric oxide synthase (eNOS), superoxide dismutase (SOD), vascular cell adhesion molecule 1 (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1) mRNA expression of human carotid artery endothelial cells.MethodsFirst, 24 apparently healthy young subjects (14 females and 10 males) performed an exercise test on cycle ergometer at low, moderate, and high intensity in which ESS where estimated from the left common carotid artery through Doppler-ultrasound for each condition. Thereafter, commercially available cultured human carotid artery endothelial cells were exposed to pulsatile flow inside a chamber system (Ibidi) for 4 hours, simulating 1 hour of exercise-induced ESS and 3 hours of resting ESS. Finally, eNOS, SOD, VCAM-1, and ICAM-1 mRNA expression were compared between groups, using GAPDH as the housekeeping gene.ResultsESS showed an intensity-dependent increment to exercise. A significant increment on eNOS mRNA expression (p < 0.05) and a significant reduction on SOD mRNA expression (p < 0.05) were observed on those cells exposed to moderate and high intensity exercise-induced ESS compared to resting ESS. No significant differences were detected on mRNA expression of VCAM-1 and ICAM-between groups.ConclusionPulsatile ESS generated during moderate and high intensity cycling might favor the upregulation of eNOS and the downregulation of SOD.

NFκB Inhibition Mitigates Serum Amyloid A-Induced Pro-Atherogenic Responses in Endothelial Cells and Leukocyte Adhesion and Adverse Changes to Endothelium Function in Isolated Aorta.

The acute phase protein serum amyloid A (SAA) is associated with endothelial dysfunction and early-stage atherogenesis. Stimulation of vascular cells with SAA increases gene expression of pro-inflammation cytokines and tissue factor (TF). Activation of the transcription factor, nuclear factor kappa-B (NFκB), may be central to SAA-mediated endothelial cell inflammation, dysfunction and pro-thrombotic responses, while targeting NFκB with a pharmacologic inhibitor, BAY11-7082, may mitigate SAA activity. Human carotid artery endothelial cells (HCtAEC) were pre-incubated (1.5 h) with 10 μM BAY11-7082 or vehicle (control) followed by SAA (10 μg/mL; 4.5 h). Under these conditions gene expression for TF and Tumor Necrosis Factor (TNF) increased in SAA-treated HCtAEC and pre-treatment with BAY11-7082 significantly (TNF) and marginally (TF) reduced mRNA expression. Intracellular TNF and interleukin 6 (IL-6) protein also increased in HCtAEC supplemented with SAA and this expression was inhibited by BAY11-7082. Supplemented BAY11-7082 also significantly decreased SAA-mediated leukocyte adhesion to apolipoprotein E-deficient mouse aorta in ex vivo vascular flow studies. In vascular function studies, isolated aortic rings pre-treated with BAY11-7082 prior to incubation with SAA showed improved endothelium-dependent vasorelaxation and increased vascular cyclic guanosine monophosphate (cGMP) content. Together these data suggest that inhibition of NFκB activation may protect endothelial function by inhibiting the pro-inflammatory and pro-thrombotic activities of SAA.

Apo A-I (Apolipoprotein A-I) Vascular Gene Therapy Provides Durable Protection Against Atherosclerosis in Hyperlipidemic Rabbits.

Gene therapy that expresses apo A-I (apolipoprotein A-I) from vascular wall cells has promise for preventing and reversing atherosclerosis. Previously, we reported that transduction of carotid artery endothelial cells with a helper-dependent adenoviral (HDAd) vector expressing apo A-I reduced early (4 weeks) fatty streak development in fat-fed rabbits. Here, we tested whether the same HDAd could provide long-term protection against development of more complex lesions. Fat-fed rabbits (n=25) underwent bilateral carotid artery gene transfer, with their left and right common carotids randomized to receive either a control vector (HDAdNull) or an apo A-I-expressing vector (HDAdApoAI). Twenty-four additional weeks of high-fat diet yielded complex intimal lesions containing lipid-rich macrophages as well as smooth muscle cells, often in a lesion cap. Twenty-four weeks after gene transfer, high levels of apo A-I mRNA (median ≥250-fold above background) were present in all HDAdApoAI-treated arteries. Compared with paired control HDAdNull-treated arteries in the same rabbit, HDAdApoAI-treated arteries had 30% less median intimal lesion volume (P=0.03), with concomitant reductions (23%-32%) in intimal lipid, macrophage, and smooth muscle cell content (P≤0.05 for all). HDAdApoAI-treated arteries also had decreased intimal inflammatory markers. VCAM-1 (vascular cell adhesion molecule-1)-stained area was reduced by 36% (P=0.03), with trends toward lower expression of ICAM-1 (intercellular adhesion molecule-1), MCP-1 (monocyte chemoattractant protein 1), and TNF-α (tumor necrosis factor-α; 13%-39% less; P=0.06-0.1). In rabbits with severe hyperlipidemia, transduction of vascular endothelial cells with an apo A-I-expressing HDAd yields at least 24 weeks of local apo A-I expression that durably reduces atherosclerotic lesion growth and intimal inflammation.

A sphingosine-1-phosphate receptor type 1 agonist, ASP4058, suppresses intracranial aneurysm through promoting endothelial integrity and blocking macrophage transmigration.

Background and PurposeIntracranial aneurysm (IA), common in the general public, causes lethal subarachnoid haemorrhage on rupture. It is, therefore, of utmost importance to prevent the IA from rupturing. However, there is currently no medical treatment. Recent studies suggest that IA is the result of chronic inflammation in the arterial wall caused by endothelial dysfunction and infiltrating macrophages. The sphingosine‐1‐phosphate receptor type 1 (S1P1 receptor) is present on the endothelium and promotes its barrier function. Here we have tested the potential of an S1P1 agonist, ASP4058, to prevent IA in an animal model.Experimental ApproachThe effects of a selective S1P1 agonist, ASP4058, on endothelial permeability and migration of macrophages across an endothelial cell monolayer were tested in vitro using a Transwell system, and its effects on the size of IAs were evaluated in a rat model of IA.Key ResultsS1P1 receptor was expressed in endothelial cells of human IA lesions and control arterial walls. ASP4058 significantly reduced FITC‐dextran leakage through an endothelial monolayer and suppressed the migration of macrophages across the monolayer in vitro. Oral administration of ASP4058 reduced the vascular permeability, macrophage infiltration and size of the IAs by acting as an S1P1 agonist in the rat model. This effect was mimicked by another two structurally‐unrelated S1P1 agonists.Conclusion and ImplicationsA selective S1P1 agonist is a strong drug candidate for IA treatment as it promotes the endothelial cell barrier and suppresses the trans‐endothelial migration of macrophages in IA lesions.

Anti-inflammatory drugs decrease infection of brain endothelial cells with EHV-1 in vitro.

Equine herpesvirus-associated myeloencephalopathy is the result of endothelial cell infection of the spinal cord vasculature with equine herpesvirus-1 (EHV-1) during cell-associated viraemia. Endothelial cell infection requires contact between infected peripheral blood mononuclear and endothelial cells. Inflammation generated during viraemia likely upregulates adhesion molecule expression on both cell types increasing contact and facilitating endothelial cell infection. Evaluating the role of anti-inflammatory drugs in decreasing endothelial cell infection with EHV-1. In vitro assay, crossover design, multiple drug testing. In vitro modified infectious centre assay using immortalised carotid artery endothelial cells or primary brain endothelial cells with plaque counts per well as outcome. Cells were either anti-inflammatory drug treated or left untreated. Significant reduction of plaque count when cells were treated compared with untreated cells. No dose-dependent effect when drug concentrations were increased to 10× dose. Treatment of both peripheral blood mononuclear cells (PBMC) and endothelial cells (EC) is required for significant plaque count reduction. In vitro study. Anti-inflammatory drugs decrease infection of endothelial cells likely by reducing contact between EHV-1 infected PBMC and endothelial cells in vitro. The role of adhesion molecules in this process needs further investigation. In vitro results suggest anti-inflammatory drug therapy during EHV-1 infection and viraemia in horses could be clinically relevant.

123 - Inhibition of the Pro-Atherogenic Action of Serum Amyloid a (SAA) by Pharmacological Blockade of NFκB Pathway

The acute phase protein serum amyloid A (SAA) is a biomarker of inflammation. Elevated circulating SAA in chronic inflammatory disorders such as diabetes mellitus is associated with endothelial dysfunction and the earliest stages of atherosclerosis. Binding of SAA to cell receptors leads to increased production of mRNA encoding tumor necrosis factor (TNF), tissue factor (TF) and cytokines such as interleukin-6 (IL-6); the proteins produced by these genes display pro-inflammatory and pro-thrombotic activities that may be involved in promoting endothelial dysfunction. Activation of the transcription factor NFκB appears to be a central canonical pathway for SAA-mediated endothelial cell dysfunction. Targeting NFκB, using the specific inhibitor BAY11-7082, may be more effective at inhibiting SAA-mediated endothelial dysfunction than a pharmaceutical cocktail that targets its receptors. Human carotid artery endothelial (HCtAE) cells were cultured to confluence under normoglycaemic (5mM glucose) or hyperglycaemic conditions (25mM glucose) conditions and pre-incubated (1.5 h; 37°C) with 10µM BAY11-7082 or vehicle (control) followed by 10μg/mL SAA (4.5 h; 37°C). Notably, TF and TNF gene expression increased markedly in HCtAE cells after SAA stimulation whereas HCtAE cells pre-treated with BAY11-7082 (with SAA) showed TF and TNF mRNA levels similar to that determined in control cells (minus SAA). In the absence of BAY11-7082, both intracellular TNF and IL-6 protein increased following incubation with SAA as determined by immunofluorescence microscopy. This SAA activity was inhibited by BAY11-7082. ELISA analysis of HCtAE cell pellets and overlying supernatant confirmed the immunofluorescence data with SAA activity inhibited by BAY11-7082; IL-6 showed a greater sensitivity to the drug than TNF. In ex-vivo leukocyte adhesion studies of mice aorta, pre-treatment with BAY11-7082 showed a significant reduction in leukocyte adhesion compared with SAA-alone treated mice. Together these data suggest that inhibition of NFκB activation may protect the endothelium from the action of SAA by inhibiting pro-inflammatory and pro-thrombotic pathways.

Evidence for functional and dynamic microcompartmentation of Cav-1/TRPV4/KCa in caveolae of endothelial cells

Ca2+-activated K+ channels (KCa) play a pivotal role in the endothelium-dependent hyperpolarization and regulation of vascular tone and blood pressure. For activation, KCa depend on an increase of intracellular calcium which is substantially mediated by Ca2+-permeable cation channels including the transient receptor potential V4 (TRPV4). It has been proposed that KCa and Ca2+-permeable cation channels may be clustered in localized positions within the cell membrane to form functional units and that caveolae may constitute the scaffolding for such microcompartmental organization. Here, we sought to elucidate the composition and functional relevance of these microcompartments in vitro and in vivo. We show that TRPV4 and small-conductance KCa2.3 are enriched in caveolae of human microvascular endothelial cells. Using immunoprecipitation, immunocytology and superresolution microscopy, we found a caveolae-dependent association between caveolin-1, TRPV4 and small conductance KCa2.3, but not intermediate conductance KCa3.1, in endothelial cells under static condition. Mechanical stimulation of cells via exposure to shear stress led to a partial de-novo colocalization of KCa3.1 with Cav-1 and TRPV4. In a mouse model of genetic Cav-1 deficiency, we found significantly reduced KCa-mediated currents as determined by patch-clamping in carotid artery endothelial cells (CAEC) from Cav-1−/− mice compared to wildtype. Functionally, Cav-1 deficiency was associated with impaired endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilation in response to shear stress and acetylcholine. In summary, our findings provide evidence for a dynamic microcompartmentation of TRPV4/KCa in caveolae of endothelial cells and highlight the importance of Cav-1 for endothelial KCa functions and flow-induced vasodilation.

Serum amyloid A receptor blockade and incorporation into high-density lipoprotein modulates its pro-inflammatory and pro-thrombotic activities on vascular endothelial cells.

The acute phase protein serum amyloid A (SAA), a marker of inflammation, induces expression of pro-inflammatory and pro-thrombotic mediators including ICAM-1, VCAM-1, IL-6, IL-8, MCP-1 and tissue factor (TF) in both monocytes/macrophages and endothelial cells, and induces endothelial dysfunction—a precursor to atherosclerosis. In this study, we determined the effect of pharmacological inhibition of known SAA receptors on pro-inflammatory and pro-thrombotic activities of SAA in human carotid artery endothelial cells (HCtAEC). HCtAEC were pre-treated with inhibitors of formyl peptide receptor-like-1 (FPRL-1), WRW4; receptor for advanced glycation-endproducts (RAGE), (endogenous secretory RAGE; esRAGE) and toll-like receptors-2/4 (TLR2/4) (OxPapC), before stimulation by added SAA. Inhibitor activity was also compared to high-density lipoprotein (HDL), a known inhibitor of SAA-induced effects on endothelial cells. SAA significantly increased gene expression of TF, NFκB and TNF and protein levels of TF and VEGF in HCtAEC. These effects were inhibited to variable extents by WRW4, esRAGE and OxPapC either alone or in combination, suggesting involvement of endothelial cell SAA receptors in pro-atherogenic gene expression. In contrast, HDL consistently showed the greatest inhibitory action, and often abrogated SAA-mediated responses. Increasing HDL levels relative to circulating free SAA may prevent SAA-mediated endothelial dysfunction and ameliorate atherogenesis.

Nilotinib Does Not Alter the Secretory Functions of Carotid Artery Endothelial Cells in a Prothrombotic or Antithrombotic Fashion.

There have been concerns about the possible prothrombotic effects of nilotinib, especially in patients having cardiovascular risk factors. The potential mechanism behind the increased risk of thromboembolic events is still not clear. In this study, we aimed to evaluate possible harmful effects of nilotinib on endothelial cells. To this aim, we examined proliferative capacity and secretory functions of healthy human carotid artery endothelial cells (HCtAECs) in response to nilotinib. 3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation method was used to determine antiproliferative effects of nilotinib on HCtAECs. The HCtAECs were incubated with 5, 10, and 100 nmol/L doses of nilotinib for 72 hours. Then, in order to assess the endothelial function, levels of nitric oxide (NO), von Willebrand factor (vWF), tissue plasminogen activator, plasminogen activator inhibitor 1 (PAI-1), and endothelin 1 (ET-1) were evaluated using enzyme-linked immunosorbent assay from tissue culture supernatants. There were slight but statistically significant decreases in cell proliferation in response to nilotinib. Nilotinib increased the secretion of t-PA, PAI-1, and vWF in a dose-dependent manner when compared with the untreated control group. The ET-1 secretion was lower in 5 nmol/L and higher in 10 and 100 nmol/L nilotinib-treated cells as compared to untreated cells. Regarding NO secretion, lower levels were observed in 5 and 10 nmol/L, and higher levels were detected in 100 nmol/L nilotinib-treated cells as compared to untreated control group cells. Considering the results obtained in our study, nilotinib does not affect the functions of endothelial cells either in a prothrombotic or an antithrombotic fashion, despite a dose-dependent decline in cell viability.

Abstract 85: MicroRNA-210 Induces Atherosclerotic Plaque Stability Through Repression of Apoptotic Pathways in Vascular Smooth Muscle Cells

Aim of the present study was to explore the role of miRNAs as potential regulators in patients with carotid artery stenosis and concordant vulnerable atherosclerotic plaques. Utilizing patient material from the Biobank of Karolinska Endarterectomies (BiKE), we were able to profile the regulation of miRNAs in patients with symptomatic vs. asymptomatic carotid stenosis undergoing carotid endarterectomy. A PCR-based miRNA array discovered 8 miRNAs (miRs-15b, -29c, -30c/d, -150, -191, -210 and -500) being substantially altered in symptomatic vs. asymptomatic patients. Next we investigated miRNA expression in 2 murine models of vascular injury and plaque vulnerability; balloon injury in Sprague-Dawley rats, and a newly-developed plaque rupture model in ApoE-/- mice, using incomplete ligation of the carotid artery with consecutive cuff injury to induce lesion rupture. In both models miR-210 was significantly repressed in response to vascular injury and plaque instability. In silico analysis revealed 48 previously validated targets of miR-210 in the vasculature. Functional miR-210 modulation analyses in both murine models revealed that the expression of three known tumorsuppressor genes (Aifm3, Apc, Casp8ap2) could be substantially altered through miR-210 modulation, affecting the proliferation rate of smooth muscle cells (SMCs) and plaque stability. By utilizing an in vitro cell streamer system, we subjected human carotid artery endothelial cells (hCAECs) and hCASMCs to laminar and oscillatory flow conditions. Interestingly, laminar flow induced miR-210 up-regulation, whereas turbulent flow suppressed miR-210 expression in both cell lines, suggesting that miR-210 repression in atherosclerotic plaques is a potential result of altered shear stress and accelerated flow patterns. Aifm3, Apc and Casp8ap2 were significantly up-regulated with disturbed flow. The present study explores the role and therapeutic potential of miRNA regulation in patients with vulnerable atherosclerotic lesions, carotid artery stenosis and increased risk of stroke. We were able to identify miR-210 as a key modulator of pathologic processes in atherosclerosis-related vascular remodelling.

The effect of solution nonideality on modeling transmembrane water transport and diffusion-limited intracellular ice formation during cryopreservation.

A new model was developed to predict transmembrane water transport and diffusion-limited ice formation in cells during freezing without the ideal-solution assumption that has been used in previous models. The model was applied to predict cell dehydration and intracellular ice formation (IIF) during cryopreservation of mouse oocytes and bovine carotid artery endothelial cells in aqueous sodium chloride (NaCl) solution with glycerol as the cryoprotectant or cryoprotective agent. A comparison of the predictions between the present model and the previously reported models indicated that the ideal-solution assumption results in under-prediction of the amount of intracellular ice at slow cooling rates (<50 K/min). In addition, the lower critical cooling rates for IIF that is lethal to cells predicted by the present model were much lower than those estimated with the ideal-solution assumption. This study represents the first investigation on how accounting for solution nonideality in modeling water transport across the cell membrane could affect the prediction of diffusion-limited ice formation in biological cells during freezing. Future studies are warranted to look at other assumptions alongside nonideality to further develop the model as a useful tool for optimizing the protocol of cell cryopreservation for practical applications.