Mouse Aortic Endothelial Cells Research Articles

Abstract 471: Influence of Platelet-specific Soluble Guanylyl Cyclase Deficiency on Atherosclerotic Plaque Formation

Introduction: The genomic locus harboring the GUCY1A3 gene has been associated with coronary artery disease (CAD) with the risk variant reducing GUCY1A3 expression via allele-specific transcription factor binding. The gene encodes the alpha 1 -subunit of the soluble guanylyl cyclase (sGC) which produces the second messenger cyclic guanosine monophosphate (cGMP) upon activation by nitric oxide, thereby influencing the function of vascular smooth muscle cells and platelets. Aim: To investigate whether a reduction in sGC expression specifically in platelets influences atherosclerotic plaque formation. Methods: By crossbreeding Pf4-Cre and sGC β 1 fl/fl mice, platelet-specific sGC knockout (PS-sGC -/- ) mice were generated. These were crossbred with Ldlr -/- mice to generate PS-sGC -/- Ldlr -/- mice. PS-sGC -/- Ldlr -/- and Ldlr -/- mice were fed a high-cholesterol diet (HCD) for 10 weeks. Serial sections of the aortic root were analyzed by Masson-Trichrome staining. Inflammatory cells in aortae were assessed using flow cytometry. In vitro adhesion assays using wildtype endothelial cells (EC) and monocytes were performed in presence of plasma of activated platelets. Results: PS-sGC -/- Ldlr -/- (n=13) mice displayed enhanced total plaque size compared to Ldlr -/- (n=15) mice (247·10 3 vs. 190·10 3 μm 2 , p<0.05). After HCD, more neutrophils (747 vs. 448, p<0.05) and Ly6C high monocytes (366 vs. 207, p<0.05) were detected in PS-sGC -/- Ldlr -/- compared to Ldlr -/- mice indicating enhanced recruitment of these cells in PS-sGC -/- Ldlr -/- mice. In in vitro adhesion assays, incubation of mouse aortic EC and monocytes with plasma from PS-sGC -/- platelets resulted in enhanced adhesion of monocytes to EC compared to plasma from wildtype platelets (13%, p<0.01). Conclusion and Outlook: Platelet-specific knockout of sGC led to increased atherosclerotic plaque formation and inflammation. In vitro adhesion assay results point to an enhanced stimulation of leukocyte adhesion to endothelial cells in PS-sGC -/- mice. Hence, factors released by platelets secondary to reduced sGC expression could mediate increased risk of CAD. Inhibiting platelet activation in carriers of the human risk variant might therefore be a promising therapeutic strategy to reduce risk of CAD.

Circulating metabolites of strawberry mediate reductions in vascular inflammation and endothelial dysfunction in db/db mice

BackgroundCardiovascular disease is 2-4-fold more prevalent in patients with diabetes. Human studies support the cardiovascular benefits of strawberry consumption but the effects of strawberry on diabetic vasculature are unknown. We tested the hypothesis that dietary strawberry supplementation attenuates vascular inflammation and dysfunction in diabetic mice. MethodsSeven-week-old diabetic db/db mice that consumed standard diet (db/db) or diet supplemented with 2.35% freeze-dried strawberry (db/db + SB) for ten weeks were compared to non-diabetic control mice (db/+). Indices of vascular inflammation and dysfunction were measured. Endothelial cells (ECs) were isolated from the vasculature to determine the influence of strawberry on them. The effect of metabolites of strawberry on endothelial inflammation was determined by incubating mouse aortic ECs (MAECs) with ±5% serum, obtained from strawberry fed mice (metabolites serum) or standard diet fed mice (control serum) ± 25 mM glucose and 100 μM palmitate. Resultsdb/db mice exhibited an increased monocyte binding to vessel, elevated blood pressure, and reduced endothelial-dependent vasorelaxation compared with db/+ mice but each defect was attenuated in db/db + SB mice. The elevation of inflammatory molecules, NOX2 and inhibitor-κB kinase observed in ECs from db/db vs. db/+ mice was suppressed in db/db + SB mice. Glucose and palmitate increased endothelial inflammation in MAECs but were normalized by co-incubation with metabolites serum. ConclusionsDietary supplementation of strawberry attenuates indices of vascular inflammation and dysfunction in diabetic db/db mice. The effect of strawberry on vasculature is endothelial-dependent and possibly mediated through their circulating metabolites. Strawberry might complement conventional therapies to improve vascular complications in diabetics.

Early life stress induces endothelial‐derived HDAC9 and ET‐1 expression

Exposure to adverse childhood experiences, also known as early life stress (ELS), is an independent risk factor for the development of cardiovascular disease (CVD) in adulthood. Endothelial dysfunction is a major contributor to the development of CVD and has been linked to childhood adversity induced CVD risk. We previously reported circulating ET‐1 levels are significantly elevated in young adults exposed to childhood adversity in humans. Endothelin‐1 (ET‐1) via the ETA receptor promotes endothelial dysfunction and vascular inflammation. However, the regulation of ET‐1 by ELS and the mechanistic link between elevated ET‐1 levels and ELS‐induced increase in CVD risk remains unknown. In a mouse model of ELS involving maternal separation and early weaning (MSEW), we previously reported that ELS induced elevated plasma ET‐1 as well as increased vascular histone deacetylase 9 (HDAC9) protein expression with no changes in expression of other HDAC isoforms. Moreover, MSEW mice develop endothelial dysfunction in adulthood that is attenuated by a pan‐inhibitor of Class I and class II HDAC proteins, trichostatin A (TSA). HDACs are a family of epigenetic enzymes that regulate transcription through deacetylation of histone and non‐histone proteins. In humans, previous studies have shown SNPs in the HDAC9 gene are prominent in large artery stroke and coronary artery disease and that upregulated HDAC9 expression is localized in endothelial cells of carotid atherosclerotic human tissue suggesting HDAC9 may play an important role in atherogenesis. Therefore, we hypothesized that vascular HDAC9 upregulation promotes endothelial dysfunction in MSEW mice via increased ET‐1. MSEW involves maternal separation 4 h/day (postnatal (PD) 2 to 5) and 8 h/day (PD6 to 16) and weaned at PD17. Normally reared litters weaned at PD21 were used as controls (CON). In adult male CON and MSEW mice, we determined aortic localization of HDAC9 and ET‐1. Immunohistolocalization showed aortic HDAC9 is upregulated specifically in the endothelial cells of MSEW mice (n=5/group). Confocal immunofluorescence imaging revealed aortic ET‐1 expression is also localized in the endothelium of both CON and MSEW mice. To examine the effects of endothelial HDAC9 on ET‐1 expression, we used mouse aortic endothelial cells (MAECs) transfected with control vector or HDAC9 plasmid incubated in the presence or absence of TSA. Cultured MAECs treated with TSA blunted ET‐1 expression (70% decrease from vehicle, p<0.05, n=3/group). Furthermore, HDAC9 over‐expression in cultured MAECs significantly increased ET‐1 expression (relative to control vector group, 1.0±0.08 vs 1.73±0.44 arbitrary units, p<0.05, n=3/group) and TSA reduced the HDAC9‐dependent ET‐1 expression (1.68 fold decrease from control vector vehicle group, p<0.05, n=3/group). Our findings indicate endothelial dysfunction previously observed in our MSEW mouse model may be mediated by HDAC9‐dependent elevated ET‐1 production. In conclusion, this study highlights the importance of ELS in the development of CVD and the implications of an epigenetic pathway as a potential mechanistic link.Support or Funding InformationNIH P01 HLP0169999; NIH K01 DK105038This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MiR-214-Dependent Increase of PHLPP2 Levels Mediates the Impairment of Insulin-Stimulated Akt Activation in Mouse Aortic Endothelial Cells Exposed to Methylglyoxal.

Evidence has been provided linking microRNAs (miRNAs) and diabetic complications, by the regulation of molecular pathways, including insulin-signaling, involved in the pathophysiology of vascular dysfunction. Methylglyoxal (MGO) accumulates in diabetes and is associated with cardiovascular complications. This study aims to analyze the contribution of miRNAs in the MGO-induced damaging effect on insulin responsiveness in mouse aortic endothelial cells (MAECs). miRNA modulation was performed by transfection of specific miRNA mimics and inhibitors in MAECs, treated or not with MGO. miRNA-target protein levels were evaluated by Western blot. PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2) regulation by miR-214 was tested by luciferase assays and by the use of a target protector specific for miR-214 on PHLPP2-3′UTR. This study reveals a 4-fold increase of PHLPP2 in MGO-treated MAECs. PHLPP2 levels inversely correlate with miR-214 modulation. Moreover, miR-214 overexpression is able to reduce PHLPP2 levels in MGO-treated MAECs. Interestingly, a direct regulation of PHLPP2 is proved to be dependent by miR-214. Finally, the inhibition of miR-214 impairs the insulin-dependent Akt activation, while its overexpression rescues the insulin effect on Akt activation in MGO-treated MAECs. In conclusion, this study shows that PHLPP2 is a target of miR-214 in MAECs, and identifies miR-214 downregulation as a contributing factor to MGO-induced endothelial insulin-resistance.

Defective p27 phosphorylation at serine 10 affects vascular reactivity and increases abdominal aortic aneurysm development via Cox-2 activation

Phosphorylation at serine 10 (S10) is the major posttranslational modification of the tumor suppressor p27, and is reduced in both human and mouse atherosclerosis. Moreover, a lack of p27-phospho-S10 in apolipoprotein E-null mice (apoE−/−) leads to increased high-fat diet-induced atherosclerosis associated with endothelial dysfunction and augmented leukocyte recruitment. In this study, we analyzed whether p27-phospho-S10 modulates additional endothelial functions and associated pathologies. Defective p27-phospho-S10 increases COX-2 activity in mouse aortic endothelial cells without affecting other key regulators of vascular reactivity, reduces endothelium-dependent dilation, and increases arterial contractility. Lack of p27-phospho-S10 also elevates aortic COX-2 expression and thromboxane A2 production, increases aortic lumen diameter, and aggravates angiotensin II-induced abdominal aortic aneurysm development in apoE−/− mice. All these abnormal responses linked to defective p27-phospho-S10 are blunted by pharmacological inhibition of COX-2. These results demonstrate that defective p27-phospho-S10 modifies endothelial behavior and promotes aneurysm formation via COX-2 activation.

Parkin Modulates ERRα/eNOS Signaling Pathway in Endothelial Cells

Background/Aims: Although a number of reports documented the important role of parkin in mitophagy, emerging evidence also indicated additional functions of parkin besides mitophagy. The present study was undertaken to investigate the role of parkin in the regulation of ERRα/eNOS pathway in endothelial cells (ECs). Methods: Mouse aortic endothelial cells (MAECs) and cardiac muscle HL-1 cells were transfected with parkin plasmid or siRNA. ERRα inhibitor XCT-790, autophagy inhibitor 3-MA and Bafilomycin A1, and caspase inhibitor Z-VAD-FMK were used to block autophagy or apoptosis. Western blotting was performed to examine the protein levels. Flow cytometry was applied to determine the cell apoptosis and ROS production. Mitochondrial membrane potential was measured using JC-1 and TMRM. Immunoprecipitation was performed to confirm the parkin effect on ERRα ubiquitination. Results: Overexpression of parkin resulted in a significant reduction of total-eNOS and p-eNOS in parallel with the downregulation of ERRα (a regulator of eNOS) protein and the enhancement of ERRα ubiquitination. To test the role of ERRα in regulating eNOS in this experimental setting, we treated ECs with ERRα inhibitor and found a decrement of total-eNOS and p-eNOS. On the contrary, overexpression of ERRα increased the levels of total-eNOS and p-eNOS. Meanwhile, parkin overexpression induced mitochondrial dysfunction and cell apoptosis in both ECs and HL-1 cells. Finally, we confirmed that the parkin effect on the regulation of eNOS was independent of the autophagy and apoptosis. Conclusion: These findings suggested that parkin overexpression downregulated eNOS possibly through the ubiquitination of ERRα in endothelial cells.

TRPV4 regulates vascular endothelial permeability during colonic inflammation in dextran sulphate sodium-induced murine colitis

The transient receptor potential vanilloid 4 (TRPV4) is a nonselective cation channel involved in physical sensing in various tissue types. The present study aimed to elucidate the function and expression of TRPV4 in colonic vascular endothelial cells during dextran sulphate sodium (DSS)-induced colitis. The role of TRPV4 in the progression of colonic inflammation was examined in the 2% DSS-induced murine colitis model using immunohistochemical analysis, Western blotting, and Evans blue dye extrusion assay. DSS-induced colitis was significantly attenuated in TRPV4-deficient (TRPV4 KO) mice when compared to wild-type mice. Repeated intrarectal administration of GSK1016790A, a TRPV4 agonist, exacerbated the severity of DSS-induced colitis. Bone marrow transfer experiments demonstrated a dominant role of TRPV4 in non-haematopoietic cells for DSS-induced colitis. DSS treatment upregulated TRPV4 expression in the vascular endothelia of colonic mucosa and submucosa. DSS treatment increased vascular permeability, which was abolished in TRPV4 KO mice. The DSS-induced increase in vascular permeability was further enhanced by intravenous administration of GSK1016790A, which was abrogated by a TRPV4 antagonist RN1734. TRPV4 was co-localized with vascular endothelial (VE)-cadherin, and VE-cadherin expression was decreased by repeated intravenous administration of GSK1016790A during colitis. Furthermore, TRPV4 activation by GSK106790A decreased VE-cadherin expression in mouse aortic endothelial cells exposed to TNF-α. These findings indicate that TRPV4 upregulation in vascular endothelial cells contributes to the progression of colonic inflammation via the activation of vascular permeability. Thus, TRPV4 is an attractive target for the treatment of inflammatory bowel diseases.

Apolipoprotein A-1 mimetic peptide 4F promotes endothelial repairing and compromises reendothelialization impaired by oxidized HDL through SR-B1

Disruption of endothelial monolayer integrity is the primary instigating factor for many cardiovascular diseases. High density lipoprotein (HDL) oxidized by heme enzyme myeloperoxidase (MPO) is dysfunctional in promoting endothelial repair. Apolipoprotein A-1 mimetic 4F with its pleiotropic benefits has been proven effective in many in vivo models. In this study we investigated whether 4F promotes endothelial repair and restores the impaired function of oxidized HDL (Cl/NO2-HDL) in promoting re-endothelialization. We demonstrate that 4F and Cl/NO2-HDL act on scavenger receptor type I (SR-B1) using human aorta endothelial cells (HAEC) and SR-B1 (-/-) mouse aortic endothelial cells. Wound healing, transwell migration, lamellipodia formation and single cell migration assay experiments show that 4F treatment is associated with a recovery of endothelial cell migration and associated with significantly increased endothelial nitric oxide synthase (eNOS) activity, Akt phosphorylation and SR-B1 expression. 4F increases NO generation and diminishes oxidative stress. In vivo, 4F can stimulate cell proliferation and re-endothelialization in the carotid artery after treatment with Cl/NO2-HDL in a carotid artery electric injury model but fails to do so in SR-B1(-/-) mice. These findings demonstrate that 4F promotes endothelial cell migration and has a potential therapeutic benefit against early endothelial injury in cardiovascular diseases.

Transient receptor potential vanilloid 4 channel regulates vascular endothelial permeability during colonic inflammation in dextran sulphate sodium-induced murine colitis.

The transient receptor potential vanilloid 4 (TRPV4) channel is a non-selective cation channel involved in physical sensing in various tissue types. The present study aimed to elucidate the function and expression of TRPV4 channels in colonic vascular endothelial cells during dextran sulphate sodium (DSS)-induced colitis. The role of TRPV4 channels in the progression of colonic inflammation was examined in a murine DSS-induced colitis model using immunohistochemical analysis, Western blotting and Evans blue dye extrusion assay. DSS-induced colitis was significantly attenuated in TRPV4-deficient (TRPV4 KO) as compared to wild-type mice. Repeated intrarectal administration of GSK1016790A, a TRPV4 agonist, exacerbated the severity of DSS-induced colitis. Bone marrow transfer experiments demonstrated the important role of TRPV4 in non-haematopoietic cells for DSS-induced colitis. DSS treatment up-regulated TRPV4 expression in the vascular endothelia of colonic mucosa and submucosa. DSS treatment increased vascular permeability, which was abolished in TRPV4 KO mice. This DSS-induced increase in vascular permeability was further enhanced by i.v. administration of GSK1016790A, and this effect was abolished by the TRPV4 antagonist RN1734. TRPV4 was co-localized with vascular endothelial (VE)-cadherin, and VE-cadherin expression was decreased by repeated i.v. administration of GSK1016790A during colitis. Furthermore, GSK106790A decreased VE-cadherin expression in mouse aortic endothelial cells exposed to TNF-α. These findings indicate that an up-regulation of TRPV4 channels in vascular endothelial cells contributes to the progression of colonic inflammation by increasing vascular permeability. Thus, TRPV4 is an attractive target for the treatment of inflammatory bowel diseases.

Abstract 21141: Cystathionine Gamma-Lyase Regulates Autophagy in Ischemic Endothelial Cells

Introduction: Peripheral arterial disease (PAD) is a chronic arterial occlusive disease leading to lower limb ischemia. Autophagy is a cell adaptive response associated with ischemic stress that may act as a double edged sword. It is essential during tissue ischemia, but may also phagocytize more organelles promoting cell death. Hydrogen sulfide (H 2 S), a gasotransmitter predominantly produced in the vasculature via cystathionine-γ-lyase (CSE), play a key regulatory role in the cardiovascular functions. However, role of H 2 S/CSE in autophagy, and its associated impact on ischemic injury is poorly understood. Objective: To investigate the role of CSE/H2S in the regulation of autophagy under ischemia using murine hind limb ischemia model and in vitro studies. Methods: Unilateral femoral artery ligation (FAL) was performed on C57BL/6J(WT) mice and tissue blood perfusion was measured using laser Doppler flowmetry. LC3B expression was measured in the skeletal muscle tissues by immunohistochemistry. We utilized mouse aortic endothelial cells (MAECs) isolated from WT and CSE deficient (CSEKO) mice respectively and immortalized them. We treated WT and CSE KO MAECs were subjected to normoxia and hypoxia with and without autophagy inhibitor bafilomycin and evaluated for LC3B. ROS levels were evaluated by DCFDH. Apoptosis was measured by LDH assay and cleaved caspase-3. Results: Compared to WT, CSEKO MAECs showed significant reduction in LC3B protein expression. Reduced LC3B and p62 mRNA expressions were also observed in CSEKO cells. In the WT mice subjected to FAL, blood perfusion was significantly reduced in the ligated limbs compared to non-ligated. Interestingly, LC3B was significantly higher in ischemic muscle compared to non-ischemic tissues. Additionally, CSE genetic deletion significantly increased ROS levels that lead to increased apoptosis. CSEKO MAECs showed increased LDH activity and elevated cleaved caspase-3 expression under hypoxia. Conclusions: Our study reveals for the first time that CSE/H 2 S regulates autophagy in ischemic endothelial cells. CSE/H 2 S may be used as a therapeutic agent to protect against ischemia injury by inhibiting autophagy during PAD.

Abstract 21290: Adipose Tissue Factors and the Adipokine Resistin-Induce Vascular Endothelial Dysfunction via Arginase Upregulation

Introduction: Obesity is a major contributor to cardiovascular disorders, for which endothelial cell (EC) dysfunction is a hallmark. Obesity-induced EC dysfunction has been linked to dysregulation of the visceral adipose tissue (VAT) resulting in increased secretion of the pro-inflammatory cytokine resistin and alterations in endothelial nitric oxide (NO) producing capacity. However, mechanisms underlying this pathology are not well understood. Hypothesis: Resistin impairs vascular EC function via upregulation of endothelial arginase1 (ARG1) expression and activity, thus limiting L-arginine availability for NO synthesis. Methods: To determine the effect of obesity on EC function, conditioned media prepared from VAT (VAT CM) of high fat diet-induced obese mice, VAT CM from lean mice or resistin (100 ηg/ml) were used to treat mouse aortic endothelial cells (MAEC) and aortic rings for 24 hr. Results: VAT CM from obese mice increased endothelial ARG1 mRNA by 34% and decreased NO level by 32% (p<0.05), vs VAT CM from lean mice. Aortic rings incubated with obese VAT CM markedly reduced endothelium-dependent vasorelaxation (EDV) to acetylcholine. Moreover, resistin levels were 143% higher in obese VAT CM compared to lean VAT CM. In MAEC, resistin increased ARG1 mRNA by 30% and decreased NO production by 39% (p<0.05). Co-treatment with the specific ARG inhibitor (ABH, 100 μM) or supplementing EC with L-arginine increased NO to near normal levels. Incubation of aortic rings with resistin also impaired EDV. This dysfunction was largely prevented by co-treatment with ABH or in aortic rings from EC specific-ARG1 knockout mice. The resistin-induced increase in ARG1 expression in MAEC is attributed to Toll-like receptor 4 (TLR4)-induced activation of p38 MAPK pathway as co-treatment with specific TLR4 inhibitor (CLI-095) inhibited p38 activation and ARG1 expression (34% and 33%, respectively, p<0.05) and increased NO production by 38% (p<0.05). Additionally, co-treatment with a specific p38 inhibitor prevented resistin-induced increase in endothelial ARG1 expression. Conclusions: VAT factors and particularly resistin induce vascular endothelial dysfunction via elevated arginase expression/activity. Support : NIH EY11766 & HL70215 & AHA fellowship

Detection and analysis of apoptosis- and autophagy-related miRNAs of mouse vascular endothelial cells in chronic intermittent hypoxia model

Endothelial dysfunction is the main pathogenic mechanism of cardiovascular complications induced by obstructive sleep apnea/hyponea syndrome (OSAHS). Chronic intermittent hypoxia (CIH) is the primary factor of OSAHS-associated endothelial dysfunction. The hypoxia inducible factor (HIF) pathway regulates the expression of downstream target genes and mediates cell apoptosis caused by CIH-induced endothelial injury. miRNAs play extensive and important negative regulatory roles in this process at the post-transcriptional level. However, the regulatory mechanism of miRNAs in CIH tissue models remains unclear. The present study established a mouse aortic endothelial cell model of CIH in an attempt to screen out specific miRNAs by using miRNA chip analysis. It was found that 14 miRNAs were differentially expressed. Of them, 6 were significantly different and verified by quantitative real-time PCR (Q-PCR), of which four were up-regulated and two were down-regulated markedly. To gain an unbiased global perspective on subsequent regulation by altered miRNAs, we established signaling networks by GO to predict the target genes of the 6 miRNAs. It was found that the 6 identified miRNAs were apoptosis- or autophagy-related target genes. Down-regulation of miR-193 inhibits CIH induced endothelial injury and apoptosis- or autophagy-related protein expression. In conclusion, our results showed that CIH could induce differential expression of miRNAs, and alteration in the miRNA expression pattern was associated with the expression of apoptosis- or autophagy-related genes.

Neonatal exposure to high oxygen levels leads to impaired ischemia-induced neovascularization in adulthood

Adverse perinatal conditions can lead to developmental programming of cardiovascular diseases. Prematurely born infants are often exposed to high oxygen levels, which in animal models has been associated with endothelial dysfunction, hypertension, and cardiac remodeling during adulthood. Here we found that adult mice that have been transiently exposed to O2 after birth show defective neovasculariation after hindlimb ischemia, as demonstrated by impaired blood flow recovery, reduced vascular density in ischemic muscles and increased tissue damages. Ischemic muscles isolated from mice exposed to O2 after birth exhibit increased oxidative stress levels and reduced expression of superoxide dismutase 1 (SOD1) and vascular endothelial growth factor (VEGF). Pro-angiogenic cells (PACs) have been shown to have an important role for postnatal neovascularisation. We found that neonatal exposure to O2 is associated with reduced number of PACs in adults. Moreover, the angiogenic activities of both PACs and mature mouse aortic endothelial cells (MAECs) are significantly impaired in mice exposed to hyperoxia after birth. Our results indicate that neonatal exposure to high oxygen levels leads to impaired ischemia-induced neovascularization during adulthood. The mechanism involves deleterious effects on oxidative stress levels and angiogenic signals in ischemic muscles, together with dysfunctional activities of PACs and mature endothelial cells.

Obesity-induced vascular inflammation involves elevated arginase activity.

Obesity-induced vascular dysfunction involves pathological remodeling of the visceral adipose tissue (VAT) and increased inflammation. Our previous studies showed that arginase 1 (A1) in endothelial cells (ECs) is critically involved in obesity-induced vascular dysfunction. We tested the hypothesis that EC-A1 activity also drives obesity-related VAT remodeling and inflammation. Our studies utilized wild-type and EC-A1 knockout (KO) mice made obese by high-fat/high-sucrose (HFHS) diet. HFHS diet induced increases in body weight, fasting blood glucose, and VAT expansion. This was accompanied by increased arginase activity and A1 expression in vascular ECs and increased expression of tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), interleukin-10 (IL-10), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) mRNA and protein in both VAT and ECs. HFHS also markedly increased circulating inflammatory monocytes and VAT infiltration by inflammatory macrophages, while reducing reparative macrophages. Additionally, adipocyte size and fibrosis increased and capillary density decreased in VAT. These effects of HFHS, except for weight gain and hyperglycemia, were prevented or reduced in mice lacking EC-A1 or treated with the arginase inhibitor 2-(S)-amino-6-boronohexanoic acid (ABH). In mouse aortic ECs, exposure to high glucose (25 mM) and Na palmitate (200 μM) reduced nitric oxide production and increased A1, TNF-α, VCAM-1, ICAM-1, and MCP-1 mRNA, and monocyte adhesion. Knockout of EC-A1 or ABH prevented these effects. HFHS diet-induced VAT inflammation is mediated by EC-A1 expression/activity. Limiting arginase activity is a possible therapeutic means of controlling obesity-induced vascular and VAT inflammation.

Autophagy Induced FHL2 Upregulation Promotes IL-6 Production by Activating the NF-κB Pathway in Mouse Aortic Endothelial Cells after Exposure to PM2.5.

Epidemiological and clinical studies have increasingly shown that fine particulate matter (PM2.5) is associated with cardiovascular morbidity and mortality, which share the common feature of PM2.5-induced vascular inflammation; however, the underlying mechanisms of how PM2.5 triggers increased inflammatory response in vascular endothelial cells are not well understood. After treating mouse aortic endothelial cells (MAECs) with different concentrations of PM2.5, we assessed interleukin (IL)-6 and four and a half LIM domains 2 (FHL2) expression in cell supernatant by enzyme-linked immunosorbent assay and Western blot, respectively, as well as activation of nuclear factor (NF)-κB and immune-response signaling pathways. Additionally, changes in pathway activation, IL-6 expression, and autophagy were evaluated under PM2.5 exposure, following FHL2 knockdown with small interfering RNA. Our results indicated that PM2.5 exposure induced FHL2 expression and IL-6 secretion, as well as activation of pathways associated with immune response. Additionally, following FHL2 knockdown, the activation of NF-κB-related pathways and IL-6 secretion was inhibited under PM2.5 exposure, although the Akt- and p38-signaling pathways were not affected. Furthermore, PM2.5 exposure induced autophagy, whereas autophagy inhibition eventually inhibited PM2.5-induced FHL2 expression. These findings suggested a novel link between autophagy induced FHL2 upregulation and IL-6 production in MAECs under PM2.5 exposure.

Quercetin reduces hydroxyurea induced cytotoxicity in immortalized mouse aortic endothelial cells.

BackgroundChronic inflammation is a characteristic of sickle cell disease (SCD), and is invariably associated with vascular endothelial injury. Hydroxyurea (HU), a naturally cytotoxic chemotherapeutic agent, is the only FDA drug approved for SCD, and is therefore naturally cytotoxic. Quercetin (QCT) is a dietary flavonoid found ubiquitously in plants and foods that have anti-oxidative and anti-inflammatory characteristics. Our hypothesis is that dietary QCT will decrease cytotoxic effects of lipopolysaccharide (LPS) and HU induced vascular cell damage.MethodsLipopolysaccharide (LPS) was used to induce inflammation in immortalized mouse aortic endothelial cells (iMAECs), providing an in vitro model of inflamed endothelial cells. The cells were exposed to LPS throughout the entire experiment. Interventions included treating the LPS exposed cells with QCT, HU, or QCT + HU over 50 hours. The 50-hour period included 24 hours of varying treatments, followed by two hours of hypoxic exposure and then 24 hours under normal aerobic exposure.ResultsLDH level was significantly higher for LPS treated versus untreated cells (P = 0.0004). LPS plus 30 micromole QCT reduced the LDH (p = 0.1, trend), whereas LPS plus 100 micromoles HU, significantly increased LDH (p = 0.0004). However, LPS plus treatment with 30 micromoles QCT/100 micromoles HU, significantly reduced LDH, compared with HU alone (p = 0.0002).DiscussionThese results suggest that quercetin may be effective against vascular endothelial cell damage for iMAECs in vitro. In particular, it shows promise in preventing HU-induced cytotoxicity, surprisingly found from these results. This latter finding is important, and should be given more consideration, since HU is the only FDA-approved drug for treating sickle cell patients, and its use is rapidly increasing.

INOS-Derived Nitric Oxide Induces Integrin-Linked Kinase Endocytic Lysosome-Mediated Degradation in the Vascular Endothelium.

ILK (integrin-linked kinase) plays a key role in controlling vasomotor tone and is decreased in atherosclerosis. The objective of this study is to test whether nitric oxide (NO) regulates ILK in vascular remodeling. We found a striking correlation between increased levels of inducible nitric oxide and decreased ILK levels in human atherosclerosis and in a mouse model of vascular remodeling (carotid artery ligation) comparing with iNOS (inducible NO synthase) knockout mice. iNOS induction produced the same result in mouse aortic endothelial cells, and these effects were mimicked by an NO donor in a time-dependent manner. We found that NO decreased ILK protein stability by promoting the dissociation of the complex ILK/Hsp90 (heat shock protein 90)/eNOS (endothelial NO synthase), leading to eNOS uncoupling. NO also destabilized ILK signaling platform and lead to decreased levels of paxillin and α-parvin. ILK phosphorylation of its downstream target GSK3-β (glycogen synthase kinase 3 beta) was decreased by NO. Mechanistically, NO increased ILK ubiquitination mediated by the E3 ubiquitin ligase CHIP (C terminus of HSC70-interacting protein), but ILK ubiquitination was not followed by proteasome degradation. Alternatively, NO drove ILK to degradation through the endocytic-lysosomal pathway. ILK colocalized with the lysosome marker LAMP-1 (lysosomal-associated membrane protein 1) in endothelial cells, and inhibition of lysosome activity with chloroquine reversed the effect of NO. Likewise, ILK colocalized with the early endosome marker EEA1 (early endosome antigen 1). ILK endocytosis proceeded via dynamin because a specific inhibitor of dynamin (Dyngo 4a) was able to reverse ILK endocytosis and its lysosome degradation. Endocytosis regulates ILK signaling in vascular remodeling where there is an overload of inducible NO, and thus its inhibition may represent a novel target to fight atherosclerotic disease.

Abstract 517: Differential Redox Profiles Due to Endothelial Heterogeneity During Oxidative Stress

Atherosclerosis, a chronic inflammatory disease characterized by plaque formation in vascular walls results in perfusion complications culminating in ischemia and induced angiogenesis. Interestingly, atherosclerosis is known largely to be an arterial disease with a preference in vascular beds, often affecting more arteries than veins. A significantly different pattern exists between the endothelial cells of these major blood vessels, highlighting the possibility of intrinsic differences between arterial and venous micro-environments including vascular endothelial cells themselves leading to variable responses during pathogenesis. Although isolated primary cells have been shown to lose some in-vivo characteristics under cell culture conditions, our data proves that these heterogeneous cell types retain the ability to differentially sense redox states. Oxidative stress is one of the key players associated with progression of atherosclerosis, marked by over-production of reactive oxygen species (ROS) and reduction in endogenous antioxidants such as glutathione (GSH). GSH, a major free radical scavenger prevents ROS damage by reducing ROS while being oxidized into glutathione disulfide (GSSG). GSSG is then recycled back to its reduced form under normal physiological conditions, hence maintaining an optimum GSH:GSSG ratio. This ratio is known to be deregulated during redox imbalance seen with oxidative stress during disease, due to decreasing GSH and increasing GSSG levels. Our experiments using Mouse Aorta Endothelial Cells (MAECs) and mouse Vena Cava Endothelial cells (VCECs) to represent arterial and venous endothelial cells display differences in this GSH:GSSG ratio as well, implicating differential redox states within these vessel types. Additionally, differences in oxidative stress related responses with regards to cell proliferation, migration, adhesion molecule expression and leukocyte recruitment in endothelial cells of arteries and veins further highlight the possibility of drastically different predispositions to atherosclerosis. Thus, endothelial cell heterogeneity between arteries and veins may lead to differential redox profiles and altered physiological functions during oxidative stress.

Abstract 212: Endothelium-Specific Deletion of Epsins Attenuates Atherosclerosis in ApoE-deficient Mouse Model Through Stabilization of IP3 Receptor 1 and Maintaining ER Homeostasis

Background: Atherosclerosis is caused in part by endothelial dysfunction due to ER stress. Understanding the mechanisms that cause ER stress and endothelial dysfunction are essential in establishing new therapies to treat atherosclerosis. Epsins are endocytic adaptor proteins that mediate the internalization of plasma membrane receptors. However, whether epsins affect atherosclerosis remains unknown. To this end, we assessed the hypothesis that endothelial epsins play a critical role in regulating atherosclerosis. Methods and Results: In an ApoE-deficient murine model, endothelial-specific double knockout mice (EC-iDKO/ApoE-/-) significantly attenuated atherosclerotic lesion and macrophage infiltration in the aortic root and arch compared to ApoE-/- mice fed Western diet for ten weeks. To elucidate underlying mechanisms, mass spectrometry analysis identified IP3R1 as one of major interactors of epsins upon exposure to oxLDL. We show that epsins bind IP3R1 and facilitate its degradation in the endothelium. Downregulation of IP3R1 by atherosclerosis promoting agents augmented ER stress in primary mouse aortic endothelial cells. In line with these findings from mouse studies, in human atherosclerotic lesion, IP3R1 is dramatically downregulated and decrease in IP3R1 expression is associated with increased ER stress. Conversely, loss of epsins stabilized IP3R1, attenuated ER stress, reduced expression of P-selectin, adhesion molecules, and chemoattractant MCP-1 in primary mouse aortic endothelial cells. Molecular mapping analysis revealed that epsin’s UIM domain is critical in facilitating the degradation of IP3R1. Delivery of a synthetic UIM peptide conjugated with atheroma-targeting Lyp-1 peptide significantly attenuated atherosclerosis in ApoE-/- animal models by stabilizing IP3R1 and maintaining ER homeostasis. Importantly, atherosclerotic lesion was restored in EC-iDKO/ApoE-/- mice by breeding them onto an endothelial-specific IP3R1 heterozygous background, suggesting that endothelial epsins promote atheroma progression in part by mediating IP3R1 degradation. Conclusions: Our data identified a novel mechanism of epsins in regulating atherosclerosis and provide a potential target to treat atherosclerosis.

Adipose Tissue Constituents and the Adipokine Resistin Impair Vascular Endothelial Function in Obesity via Elevated Arginase Activity

ObjectiveDefine the molecular mechanisms underlying obesity‐induced endothelial dysfunction with an emphasis on the role of arginase (ARG) as a target for the better management of disease progression.IntroductionArginase (ARG) is expressed in endothelial cells (EC) in 2 isoforms (ARG1 and ARG2) and can contribute to vascular and endothelial dysfunction by limiting arginine availability for nitric oxide (NO) synthesis. Obesity is an important factor in several cardiovascular comorbidities and is associated with vascular endothelial dysfunction. Obesity is characterized by adipose tissue dysregulation resulting in increased secretion of pro‐inflammatory cytokines and adipokines such as resistin, which induces insulin resistance and endothelial dysfunction. Mechanisms underlying adipose tissue dysregulation‐induced endothelial dysfunction is not well understood.MethodsTo determine the effect of obesity on adipocyte dysregulation, mature adipocytes were extracted from visceral adipose tissues of diet‐induced obese (DIO) and lean mice to determine gene expression of pro‐inflammatory cytokines and adipokines. Additionally, Mouse aortic endothelial cells (MAEC) and aortic rings from lean mice were exposed to conditioned media (CM) prepared from visceral adipose tissues of DIO and lean mice or resistin (100 ηg/ml) for 24 hr to assess endothelial and vascular functions.ResultsAdipocytes from DIO mice showed increased mRNA expression of the pro‐inflammatory cytokines (TNF‐α and MCP‐1) and the adipokine resistin, compared to lean mice cells. MAEC exposed to CM from DIO mice showed increased mRNA expression of ARG1 and ARG2 (34% and 77%, respectively, p<0.05) accompanied by decreased NO production by 32%, p<0.01. Aortic rings incubated with CM from DIO mice exhibited a marked reduction in endothelium‐dependent vasorelaxation (EDV) to acetylcholine and increased contractile responses to phenylephrine (PE). Moreover, CM from DIO mice caused a 143% increase in resistin levels compared to CM from lean mice. Exposure of MAEC to resistin resulted in increased expression of ARG1 mRNA and decreased NO production (30% and 39%, respectively, p<0.05). Exposure of aortic rings to resistin impaired EDV. These dysfunctions were largely prevented by co‐treatment with the specific ARG inhibitor ABH (100 μM). The resistin‐induced increase in ARG1 expression in MAEC involves activation of the p38 mitogen‐activated protein kinase pathway as co‐treatment with p38 inhibitor (SB202190) prevented both effects in response to resistin.ConclusionAdipose tissue constituents from DIO mice and the pro‐inflammatory adipokine resistin impair EC‐dependent vasorelaxation via elevated arginase expression/activitySupport or Funding InformationNIH Grant EY11766 & NIH HL70215