Endothelial Autophagy Research Articles

Altered mitochondrial dynamics contributes to endothelial dysfunction in hypertension

AimsImpaired mitochondrial dynamics is implicated in cardiovascular diseases. Alterations in mitochondrial dynamics and reactive oxygen species production have been shown to be associated with the development of hypertension. However, the role of mitochondrial dynamics in endothelial dysfunction during the development of hypertension remains unknown.MethodsEffects of mitochondrial fission inhibitor mdivi‐1 on vascular contraction and relaxation were examined on mesenteric arteries isolated from WKY or SHR rats. Mitochondrial networks in aortic endothelia were assessed using transmission electron microscope, and further evaluated with laser confocal after labeling with 100 nmol/L MitoTracker Green FM. In vitro, effects of Angiotensin□(10−6 M, 24 h) on mitochondrial morphology in HUVECs were investigated.ResultsPretreatment with mdivi‐1 inhibited PE‐induced mesenteric artery constriction in WKY rats, and the inhibitory effect of mdivi‐1 was attenuated on endothelium‐denuded mesenteric arteries. Then we observed mitochondrial fragmentation, increased mitochondrial reactive oxygen species production and increased expression of dynamin‐related protein 1 (Drp1) in aortic endothelial cells from SHR rats. Mdivi‐1 pretreatment ameliorated acetylcholine induced mesenteric artery relaxation in SHR rats. In cultured HUVECs exposed to Angiotensin□, we observed a similar loss of mitochondrial networks and increased expression of Drp1. Altered mitochondrial dynamics was associated with increased mitochondrial reactive oxygen species production and a marked impairment of insulin‐stimulated activation of endothelial nitric oxide synthase and endothelial autophagy. Silencing Drp1 with siRNA blunted Angiotensin□induced alterations in mitochondrial networks, reactive oxygen species production, endothelial nitric oxide synthase activation and endothelial autophagy.ConclusionsThese findings show that up‐regulation of Drp1 during hypertension exacerbates endothelial dysfunction by increasing mitochondrial reactive oxygen species and subsequently inhibiting endothelial nitric oxide synthase activation and autophagy.Support or Funding InformationThis study was supported by the grants from the National Natural Science Foundation of China (Grant Nos. AWS16J018, 81670253)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Arterial dysfunction displayed by old mice with repressed endothelial cell autophagy is rescued by pharmacological activation of purinergic 2Y1 receptors

Earlier we reported that genetic repression of endothelial cell (EC) autophagy impairs autocrine signaling via the purinergic 2Y1 (P2Y1) receptor to EC nitric oxide (NO) synthase (eNOS), and prevents NO generation in response to shear stress. Herein we sought to determine whether aging‐associated endothelial dysfunction displayed by mice with defective vascular autophagy could be restored by promoting purinergic signaling. First, mRNA and protein expression of autophagy markers was lower in ECs and arterial homogenates of 22–23 month old (old) vs. 6–7 month old (adult) mice. Second, the ability of aged mice with repressed basal autophagy to upregulate this process in response to physiological stimuli was prevented. Specifically, 14 h fasting and 60‐min treadmill‐running increased autophagy in arteries from adult but not old mice. Third, autophagy suppression (3‐methyladenine) impaired flow‐mediated vasodilation in arteries from adult mice evaluated ex vivo, while autophagy activation (trichostatin‐A) preserved vasodilation in arteries from older animals. Finally, the selective P2Y1 receptor agonist 2‐methylthio‐ADP restored arterial function in arteries from aged mice whereas P2Y1 receptor inhibition using MRS2179 precipitated an aging phenotype in vessels from adult animals. We provide the first evidence that aging : (i) prevents the autophagic response to fasting and acute exercise and (ii) attenuates flow‐mediated vasodilation in a manner that can be restored by intervening distal to defective vascular autophagy i.e., by pharmacological promotion of purinergic autocrine signaling. Further research concerning the G protein‐coupled P2Y family of receptors is warranted in an effort to explore new therapeutic treatment options for aging‐related endothelial dysfunction that is secondary to defective EC autophagy.Support or Funding InformationAHA 16GRNT31050004, NIH RO3AGO52848, Seed Grants from the UU Office of the Vice‐President for Research, the UU College of Health, the UU Center on Aging, the UU Diabetes and Metabolism Center, and the Diabetes Research Center at Washington University at Saint Louis 5 P30 DK020579 (JDS)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Antioxidant role of autophagy in maintaining the integrity of glomerular capillaries

ABSTRACT Autophagy is a lysosomal degradation system by which cytosolic materials and damaged organelles are broken down into basic components. To explore the physiological role of autophagy in glomerular endothelial cells (GEnCs), we compared the autophagic flux among cells in the kidney under starvation. Inhibition of autophagy by chloroquine administration significantly increased the number of autophagosomes or autolysosomes in GEnCs and proximal tubular cells, but not in podocytes, suggesting that the GEnCs exhibit substantial autophagic activity. Next, we analyzed endothelial and hematopoietic cell-specific atg5-deficient mice (atg5-conditional KO [cKO] mice). Glomeruli of 4-wk-old atg5-cKO mice exhibited slightly distended capillary loops accompanied by an accumulation of reactive oxygen species (ROS). Glomeruli of 8-wk-old atg5-cKO mice showed a lobular pattern with thickening of the capillary loops and mesangial matrix expansion; however, the vasculature of other organs was preserved. The atg5-cKO mice died by 12 wk of age, presumably due to pancytopenia resulting from the defect in their hematopoietic lineages. Therefore, we subjected 4-wk atg5-cKO mice to irradiation followed by bone marrow transplantation from normal littermates. Transplanted mice recapitulated the glomerular phenotypes of the atg5-cKO mice with no obvious histological changes in other organs. Twelve-mo-old transplanted mice developed mesangiolysis and glomerulosclerosis with significant deterioration of kidney function. Administration of N-acetyl-l-cysteine, a ROS scavenger, to atg5-cKO mice rescued the glomerular phenotypes. These data suggest that endothelial autophagy protects glomeruli from oxidative stress and maintains the integrity of glomerular capillaries. Enhancing endothelial autophagy may provide a novel therapeutic approach to minimizing glomerular diseases.

Mechanisms of autophagy activation in endothelial cell and their targeting during normothermic machine liver perfusion.

Ischaemia-reperfusion injury (IRI) is the leading cause of injury seen in the liver following transplantation. IRI also causes injury following liver surgery and haemodynamic shock. The first cells within the liver to be injured by IRI are the liver sinusoidal endothelial cells (LSEC). Recent evidence suggests that LSEC co-ordinate and regulates the livers response to a variety of injuries. It is becoming increasingly apparent that the cyto-protective cellular process of autophagy is a key regulator of IRI. In particular LSEC autophagy may be an essential gatekeeper to the development of IRI. The recent availability of liver perfusion devices has allowed for the therapeutic targeting of autophagy to reduce IRI. In particular normothermic machine liver perfusion (NMP-L) allow the delivery of pharmacological agents to donor livers whilst maintaining physiological temperature and hepatic flow rates. In this review we summarise the current understanding of endothelial autophagy and how this may be manipulated during NMP-L to reduce liver IRI.

MicroRNA-100 Suppresses Chronic Vascular Inflammation by Stimulation of Endothelial Autophagy.

The interaction of circulating cells within the vascular wall is a critical event in chronic inflammatory processes, such as atherosclerosis, but the control of the vascular inflammatory state is still largely unclear. This study was undertaken to characterize the function of the endothelial-enriched microRNA miR-100 during vascular inflammation and atherogenesis. Based on a transcriptome analysis of endothelial cells after miR-100 overexpression, we identified miR-100 as a potent suppressor of endothelial adhesion molecule expression, resulting in attenuated leukocyte-endothelial interaction in vitro and in vivo as shown by flow cytometry and intravital imaging. Mechanistically, miR-100 directly repressed several components of mammalian target of rapamycin complex 1-signaling, including mammalian target of rapamycin and raptor, which resulted in a stimulation of endothelial autophagy and attenuated nuclear factor κB signaling in vitro and in vivo. In a low-density lipoprotein receptor-deficient atherosclerotic mouse model, pharmacological inhibition of miR-100 resulted in enhanced plaque lesion formation and a higher macrophage content of the plaque, whereas a systemic miR-100 replacement therapy had protective effects and attenuated atherogenesis, resulting in a decrease of plaque area by 45%. Finally, analysis of miR-100 expression in >70 samples obtained during carotid endarterectomy revealed that local miR-100 expression was inversely correlated with inflammatory cell content in patients. In summary, we describe an anti-inflammatory function of miR-100 in the vascular response to injury and inflammation and identify an important novel modulator of mammalian target of rapamycin signaling and autophagy in the vascular system. Our findings of miR-100 as a potential protective anti-athero-miR suggest that the therapeutic replacement of this microRNA could be a potential strategy for the treatment of chronic inflammatory diseases, such as atherosclerosis, in the future.

Endothelial autophagy as a key mechanism in arterial diseases

Blood flow imposes shear stress on endothelial cells (ECs). ECs are able to convert these mechanical stimuli into intracellular signals that affect cellular function. As deregulated autophagy is associated with an acceleration of a variety of cardiovascular and metabolic diseases where impaired flow-mediated EC responses promote cardiovascular risk, we hypothesized that endothelial autophagy and endothelial function interact.We found that defective endothelial autophagy, caused by targeted deletion of the Atg5 gene in ECs alone, results in selective loss of flow-induced vaso-dilation in mesenteric arteries and in kidneys ex vivo; this leads to increased cerebral and renal vascular resistance in vivo. Furthermore, we find a crucial pathophysiological role for endothelial autophagy in flow-mediated outward arterial remodelling, prevention of neointima formation following wire injury and recovery after myocardial infarction. Together, these findings unravel a fundamental role for autophagy in endothelial function, linking cell proteostasis to mechanosensing signaling that will be discussed. These findings also open new questions regarding the potential role of endothelial proteostasis in arterial diseases.

Endothelial cell autophagy in chronic intermittent hypoxia is impaired by miRNA-30a-mediated translational control of Beclin-1

// Kai-Xiong Liu 1, 2, 3 , Qin Chen 4 , Gong-Ping Chen 1, 2, 3 , Jian-Chai Huang 1, 2, 3 , Jie-Feng Huang 1, 2, 3 , Xin-Ru He 1, 2, 3 , Ting Lin 1, 2, 3 and Qi-Chang Lin 1, 2, 3 1 Department of Respiratory Disease, The First Affiliated Hospital of Fujian Medical University, Fuzhou 350005, China 2 Laboratory of Respiratory Disease of Fujian Medical University, Fuzhou 350005, China 3 Fujian Provincial Sleep-Disordered Breathing Clinic Center, Fuzhou 350005, China 4 Integrated Chinese and Western Medicine Colleges, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, China Correspondence to: Qi-Chang Lin, email: chang4e@126.com Keywords: chronic intermittent hypoxia (CIH); obstructive sleep apnea (OSA); autophagy; Beclin-1; miR-30a Received: August 21, 2017 Accepted: September 03, 2017 Published: January 02, 2018 ABSTRACT Chronic intermittent hypoxia (CIH) in obstructive sleep apnea causes damages of aortic endothelial cells, which predisposes development of many cardiovascular diseases. Recently, both altered expression of microRNAs (miRNAs) and impaired autophagy were found to be associated with endothelial cell dysfunction in CIH. However, the exact molecular regulatory pathway has not been determined. Here, we addressed this question. In a mouse model of CIH, we detected significant upregulation of miR-30a, a miRNA that targets 3′-UTR of autophagy-associated protein 6 (Beclin-1) mRNA to suppress the protein translation, which subsequently attenuated the endothelial cell autophagy against cell death. Indeed, unlike Beclin-1 mRNA, the Beclin-1 protein in endothelial cells did not increase after CIH. Suppression of miR-30a by expression of antisense of miR-30a significantly increased Beclin-1 levels to enhance endothelial cell autophagy in vitro and in vivo , which improved endothelial cell survival against CIH. Together, these data suggest that endothelial cell autophagy in CIH may be attenuated by miR-30a-mediated translational control of Beclin-1, as an important cause of endothelial cell dysfunction and damage.

Cytokine-induced autophagy promotes long-term VCAM-1 but not ICAM-1 expression by degrading late-phase I\u03baB\u03b1

Pro-inflammatory cytokines are known to induce endothelial cell autophagy, but the role of autophagy in regulating the expression of pro-inflammatory molecules has not been characterized. We hypothesized that autophagy facilitates expression of endothelial adhesion molecules. TNFα and IL-1β induced autophagy markers in human umbilical vein endothelial cells and inhibition of autophagy by 3-methyladenine (3-MA) blocked adhesion of Jurkat lymphocytes. Interestingly, 3-MA suppressed VCAM-1 but not ICAM-1 expression at 24 hours but not 6 hours. 3-MA suppressed VCAM-1 transcription and decreased nuclear NF-κB p65 level at 6 hours but not at 2 hours. Cytokines induced a biphasic degradation of IκBα and 3-MA selectively blocked the late-phase IκBα degradation. Our results suggest that cytokine-induced autophagy contributes to late-phase IκBα degradation, facilitates NF-κB nuclear translocation and VCAM-1 transcription for long-term VCAM-1 expression. With a cytokines array assay, we found that 3-MA also inhibited IP-10 expression. These findings provide new information about the role of endothelial autophagy in persistent expression of VCAM-1 and IP-10 which enhance lymphocyte recruitment and adhesion to endothelium.

Autophagy is required for endothelial cell alignment and atheroprotection under physiological blood flow

It has been known for some time that atherosclerotic lesions preferentially develop in areas exposed to low SS and are characterized by a proinflammatory, apoptotic, and senescent endothelial phenotype. Conversely, areas exposed to high SS are protected from plaque development, but the mechanisms have remained elusive. Autophagy is a protective mechanism that allows recycling of defective organelles and proteins to maintain cellular homeostasis. We aimed to understand the role of endothelial autophagy in the atheroprotective effect of high SS. Atheroprotective high SS stimulated endothelial autophagic flux in human and murine arteries. On the contrary, endothelial cells exposed to atheroprone low SS were characterized by inefficient autophagy as a result of mammalian target of rapamycin (mTOR) activation, AMPKα inhibition, and blockade of the autophagic flux. In hypercholesterolemic mice, deficiency in endothelial autophagy increased plaque burden only in the atheroresistant areas exposed to high SS; plaque size was unchanged in atheroprone areas, in which endothelial autophagy flux is already blocked. In cultured cells and in transgenic mice, deficiency in endothelial autophagy was characterized by defects in endothelial alignment with flow direction, a hallmark of endothelial cell health. This effect was associated with an increase in endothelial apoptosis and senescence in high-SS regions. Deficiency in endothelial autophagy also increased TNF-α-induced inflammation under high-SS conditions and decreased expression of the antiinflammatory factor KLF-2. Altogether, these results show that adequate endothelial autophagic flux under high SS limits atherosclerotic plaque formation by preventing endothelial apoptosis, senescence, and inflammation.

Abstract 208: Excessive Autophagy Contributes to Microvascular Endothelial Dysfunction in Prehypertensive Spontaneously Hypertensive Rats

Autophagy exists in vascular endothelial cells, however, the exact role of autophagy in hypertensive-associated vascular endothelial dysfunction remains largely elusive. We assessed the hypothesis that autophagy is one of the mechanisms of endothelial dysfunction in normotensive spontaneously hypertensive rats (SHRs). Age-matched male SHRs and Wistar Kyoto rats (WKY) aged 4 weeks (young) and 12 weeks (adult) were randomized into 2 groups and treated daily for 2 weeks by a) gavage with vehicle alone (normal saline) or b) intraperitoneally injection of rapamycin (1 mg/kg/day), and the vascular function of their isolated aorta and mesenteric artery was assessed in vitro . Furthermore, human umbilical vein endothelial cells (HUVECs) were incubated serum-starved for 6 h to induce excessive cell autophagy, and then incubated with DMEM containing 10% FBS and a) treated with 10 nmol/L insulin-like growth factor 1 (IGF-1) for 12 h, b) treated with 100 nmol/L Rapamycin for 1 h for 37°C. Compared with WKY, young and adult SHRs showed endothelial dysfunction of the aorta and mesenteric artery, along with decreased phosphorylated (p)-Akt, p-mTOR, and autophagic marker protein p62 and increased LC3 II/I in microvascular but not aortic tissues. Treatment of young SHRs with the mTOR inhibitor rapamycin aggravated the endothelial autophagy and subsequent impaired endothelial function. Moreover, IGF-1 significantly activated Akt/mTOR signaling in HUVECs and reduced autophagic levels, while rapamycin inhibited p-mTOR and increased autophagic levels. In conclusion, mesenteric endothelial dysfunction in prehypertensive SHRs is at least partly attributable to excessive autophagy in vascular tissues, and is likely mediated by the underactive Akt/mTOR signaling pathway; however, autophagy appears not to be responsible for the macrovascular endothelial dysfunction.

Abstract 590: Autophagy is Required for Endothelial Atheroprotective Signaling Under Physiological Flow

Objective: Preferential development of atherosclerotic lesions in areas of low shear stress is associated with increased endothelial inflammation, apoptosis and senescence. On the contrary, high shear stress areas are protected from plaque development, but the mechanisms remain elusive. Autophagy is a protective mechanism allowing recycling of defective organelles and proteins to maintain cellular homeostasis. Our aim was to understand the role of autophagy in athero-protective effects of high shear stress. Approach and Results: We used the parallel plate chamber system in vitro to generate different shear stress conditions on endothelial cell deficient or not in autophagy (shRNA Atg5 vs. control) and examined autophagic flux in cells transfected with RFP-GFP-LC3 plasmid. Endothelial autophagy was silenced in Atg5 flox/flox ; VE-cadherin-cre mice. We first demonstrated in human and murine arteries and in cultured endothelial cells that atheroprotective shear stress activates endothelial autophagic flux. On the opposite, endothelial cells exposed to atheroprone low shear stress displayed inefficient autophagy, associated with activation of mTOR, inhibition of AMPKα pathways and blockade of the autophagic flux. Interestingly, plaque burden augmented specifically in areas usually atheroresistant in ApoE -/- hypercholesterolemic mice deficiency in endothelial autophagy. Both in cultured endothelial cells and in transgenic mice, deficiency in endothelial autophagy was associated with a defect in endothelial alignment with flow direction, a hallmark of endothelial cell health. Deficiency in endothelial autophagy increased endothelial senescence and TNFα-induced inflammation under high shear stress conditions. These effects were associated with impaired KLF2 activation. In transgenic mice, endothelial senescence and apoptosis was augmented in high shear stress areas of the descending thoracic aorta when compared to control littermate mice. Conclusions: Altogether, these results show that adequate endothelial autophagic flux under high shear stress limits atherosclerotic plaque formation by preventing inflammation, senescence and apoptosis.

321 - Activation of endothelial autophagy is required for endothelial alignment in flow and atheroprotective signaling

321 - Activation of endothelial autophagy is required for endothelial alignment in flow and atheroprotective signaling

TNF-\u03b1 stimulates endothelial palmitic acid transcytosis and promotes insulin resistance

Persistent elevation of plasma TNF-α is a marker of low grade systemic inflammation. Palmitic acid (PA) is the most abundant type of saturated fatty acid in human body. PA is bound with albumin in plasma and could not pass through endothelial barrier freely. Albumin-bound PA has to be transported across monolayer endothelial cells through intracellular transcytosis, but not intercellular diffusion. In the present study, we discovered that TNF-α might stimulate PA transcytosis across cardiac microvascular endothelial cells, which further impaired the insulin-stimulated glucose uptake by cardiomyocytes and promoted insulin resistance. In this process, TNF-α-stimulated endothelial autophagy and NF-κB signaling crosstalk with each other and orchestrate the whole event, ultimately result in increased expression of fatty acid transporter protein 4 (FATP4) in endothelial cells and mediate the increased PA transcytosis across microvascular endothelial cells. Hopefully the present study discovered a novel missing link between low grade systemic inflammation and insulin resistance.

Endothelial-specific deletion of autophagy-related 7 (ATG7) attenuates arterial thrombosis in mice

BackgroundThrombosis persists as a leading cause of morbidity and mortality. Given that endothelial cells (ECs) play a central role in regulating thrombosis, understanding the molecular endothelial cues that regulate susceptibility or resistance to thrombosis have important translational implications. Accordingly, we evaluated the role of endothelial autophagy in the development of thrombosis. MethodsWe generated mice in which the essential autophagy-related 7 (ATG7) gene was conditionally deleted from ECs (EC-ATG7−/− mice). Three in vivo models of thrombosis were used, and mechanistic studies were conducted with cultured human umbilical vein endothelial cells (HUVECs). ResultsWe silenced ATG7 in HUVECs and observed >60% decreases in tumor necrosis factor (TNF)-α–induced tissue factor (TF) transcript levels, protein expression, and activity. TF mRNA levels in the carotid arteries of EC-ATG7−/− mice subjected to the prothrombotic stimulus FeCl3 were lower than those in the similarly treated wild-type (WT) littermate group. Compared with WT mice, EC-ATG7−/− mice exhibited prolonged time to carotid (2-fold greater) and mesenteric (1.3-fold greater) artery occlusion following FeCl3 injury. The thrombi generated in laser-injured cremasteric arterioles were smaller in EC-ATG7−/− mice compared with WT mice, and took 2.3-fold longer to appear. ConclusionsTaken together, these results provide definitive evidence that loss of endothelial ATG7 attenuates thrombosis and reduces the expression of TF. Our findings demonstrate that endothelial ATG7, and thus autophagy, is a critical and previously unrecognized target for modulating the susceptibility to thrombosis.

Ox-LDL-Induced MicroRNA-155 Promotes Autophagy in Human Endothelial Cells via Repressing the Rheb/ mTOR Pathway

Background/Aims: Autophagy, an evolutionary conserved biological process, is activated in cells to cope with various types of stress. MicroRNAs control several activities related to autophagy. However, the role of autophagy-related microRNAs during atherosclerosis is far from known. MicroRNA-155 was identified to be a crucial regulator of atherosclerosis. The objectives of the study were to analyze the effect of microRNA-155 on autophagic signaling and explore its mechanism in human endothelial cells under ox-LDL stress. Methods: The study included human endothelial cells surrogate EA.hy926 lines (EA.hy926 cells). The expression of microRNA-155 was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The effect of microRNA-155 on endothelial autophagy was observed along with the expression levels of Rheb, LC3B, Beclin1, and P62/SQSTM1 by western blotting (WB) and immunofluorescence through microRNA-155 overexpression or inhibition. Bioinformatics analysis and Luciferase reporter assay were used to explore the target gene of microRNA-155. Cell viability and apoptosis were examined by 3-[4,5-dimethylthiazol-2-yl]-5- [3-carboxy-methoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium inner salt (MTS) assay and TdT-mediated dUTP Nick-End Labeling (TUNEL) apoptosis assay. Results: MicroRNA-155 expression was significantly increased under ox-LDL stress. MicroRNA-155 increased autophagic activity, while inhibition of it alleviated ox-LDL-induced autophagy in EA.hy926 endothelial cells. In addition, dual-luciferase reporter assays showed that microRNA-155 suppressed Rheb transcription. MicroRNA-155 increased autophagic activity in EA.hy926 cells via inhibition of Rheb-mediated mTOR/P70S6kinase/4EBP signaling pathway. Furthermore, we demonstrated that microRNA-155 could regulate not only autophagy but also apoptosis in EA.hy926 cells. Conclusions: MicroRNA-155 works as a regulator of endothelial function under ox-LDL stress, making it a potential candidate for the novel therapeutic strategies against atherosclerotic diseases.

0516 : Shear stress regulates endothelial autophagy: consequences on endothelial senescence and atherogenesis

Atherosclerotic plaques form preferentially in arterial areas exposed to low shear stress (LSS) where endothelial cells express senescence-associated phenotypes. We tested the hypothesis that endothelial autophagy is an anti-senescence and anti-atherogenic process regulated by shear stress. Endothelial cells exposure to LSS (2 dyn/cm 2 , 24h) decreased LC3II/I ratio compared to high shear stress (HSS, 20 dyn/cm 2 ) (1.6±0.2 vs. 2.0±0.3, respectively p<0.01). Bafilomycin A1 revealed that autophagic flux was elevated in HSS conditions. In mouse aorta, the surface of LC3 punctae/cell was lower in LSS than in HSS areas (9±1 vs. 15±1 mm 2 /cell, respectively p=0.01). Similar results were obtained in human carotid arteries. Both mTOR and AMPK pathways were involved in autophagy regulation by shear stress. Using culture endothelial cells, we observed more senescence-associated β-galactosidase activity under LSS than under HSS (48±4 vs. 37±3% positive cells, respectively p<0.05). Autophagy inhibition using wortmannin under HSS increased endothelial senescence when compared to vehicle (71±7% vs. 37±4% positive cells, respectively p<0.01), whereas autophagy activation using rapamycin under LSS prevented endothelial senescence (34±1 vs. 48±4% positive cells, respectively p<0.05). In vivo , endothelial senescence in HSS areas was higher in mice with an endothelial-specific deletion of Atg5 than in control mice, as evaluated by SA-β-gal staining (3.8±0,8 vs. 0.9±0,1 positive cells/mm 2 , respectively p<0.01). No difference was observed in LSS areas. ApoE -/- mice with an endothelial specific deletion of Atg5 developed larger atherosclerotic lesions in HSS areas than control mice (3.1±0.5 vs. 1.4±0.4% respectively p<0.05), but no difference was observed in LSS areas. In conclusion, endothelial autophagy is defective in endothelial cells in LSS areas and is associated with cellular senescence. This seems to contribute to the preferential development of atherosclerotic lesions in these areas. The author hereby declares no conflict of interest

The Protective Role of Autophagy in Matrix Metalloproteinase-Mediated Cell Transmigration and Cell Death in High-Glucose-Treated Endothelial Cells.

Diabetes mellitus may cause vascular endothelial damage via endothelial matrix metalloproteinase-2 (MMP-2). The role of endothelial autophagy in MMP-2-mediated cell injury in response to high-glucose (HG) stimulation was rarely described. In this study, we used HG-treated human umbilical vein endothelial cells (HUVECs) to investigate the effect of autophagy on MMP-2-induced cell transmigration and apoptosis. THP-1 transmigration was detected by the transmigration assay. Light chain 3 (LC3, representing autophagy), MMP-2, and poly (ADP-ribose) polymerase (PARP, representing apoptosis) of HG (33mM)-treated HUVECs were evaluated by western blot analysis. The MMP-2 activity was also examined by gelatin zymography. We used GM6001 (10μM, an MMP-2 inhibitor) to investigate the relationship of MMP-2 and THP-1 transmigration. Using 3-methyladenine (3MA, 5mM, an LC3 inhibitor), we explored the effects of autophagy on MMP-2 expression, THP-1 transmigration, and apoptosis. Our results showed that HG increased LC3-II expression, MMP-2 activity, THP-1 transmigration, and cleaved PARP expression in a time-dependent manner (0-48h); among them, LC3-II appeared earlier (0-24h) than the others (24-48h). GM6001 suppressed MMP-2 activity and ameliorated THP-1 transmigration. 3MA suppressed LC3-II expression and increased MMP-2 expression, THP-1 transmigration, and cleaved PARP expression. From these sequential findings, we demonstrated that autophagy plays a protective role in MMP-2-mediated cell transmigration and cell death in HG-stimulated HUVECs.

Intact endothelial autophagy is required to maintain vascular lipid homeostasis.

The physiological role of autophagic flux within the vascular endothelial layer remains poorly understood. Here, we show that in primary endothelial cells, oxidized and native LDL stimulates autophagosome formation. Moreover, by both confocal and electron microscopy, excess native or modified LDL appears to be engulfed within autophagic structures. Transient knockdown of the essential autophagy gene ATG7 resulted in higher levels of intracellular (125) I-LDL and oxidized LDL (OxLDL) accumulation, suggesting that in endothelial cells, autophagy may represent an important mechanism to regulate excess, exogenous lipids. The physiological importance of these observations was assessed using mice containing a conditional deletion of ATG7 within the endothelium. Following acute intravenous infusion of fluorescently labeled OxLDL, mice lacking endothelial expression of ATG7 demonstrated prolonged retention of OxLDL within the retinal pigment epithelium (RPE) and choroidal endothelium of the eye. In a chronic model of lipid excess, we analyzed atherosclerotic burden in ApoE(-/-) mice with or without endothelial autophagic flux. The absence of endothelial autophagy markedly increased atherosclerotic burden. Thus, in both an acute and chronic invivo model, endothelial autophagy appears critically important in limiting lipid accumulation within the vessel wall. As such, strategies that stimulate autophagy, or prevent the age-dependent decline in autophagic flux, might be particularly beneficial in treating atherosclerotic vascular disease.

Abstract 10824: A Novel Role of Endothelial Autophagy in the Regulation of Thrombosis in vivo

Background: Arterial thrombosis of native and graft vessels is the inciting event for acute coronary syndromes, and the cause of significant cardiovascular morbidity and mortality. Endothelial cell (EC) dysfunction is believed to play a central role in the pathobiology of thrombosis. ECs are activated upon atherosclerotic plaque rupture and induce the expression of tissue factor (TF) with resultant atherothrombotic sequelae. The molecular basis of how endothelial TF is unregulated remains unknown. We hypothesize that endothelial autophagy, a process involved in cellular homeostasis and stress responses, plays a causal role in the expression of TF, and via this mechanism modulates thrombosis. Methods: We generated EC-specific ATG7 knockout (EC-ATG7 -/- ) mice using CreLoxP technology. We used small interfering RNA (siRNA) to knockdown and characterize the effect of ATG7 inhibition in HUVECs. We also employed intravital microscopy in FeCl 3 -induced carotid and laser-induced cremaster artery injury to evaluate thrombosis. Results: siRNA-mediated knockdown of ATG7 in HUVEC resulted in decreased TF mRNA and protein expression by over 60% (N=3-5; p&lt;0.05). Mean TF mRNA level in EC-ATG7 -/- mice was 43% lower (p&lt;0.05) compared with WT mice, after FeCl 3 -induced carotid artery injury. Time to carotid artery occlusion was significantly prolonged in EC-ATG7 -/- mice compared with WT mice (15.5±2.0 and 31.0±4.0 min, respectively; N=10/group; p=0.003). The time to maximum platelet fluorescence intensity in EC-ATG7 -/- mice was significantly prolonged relative to that for WT mice (1.0±0.6 and 2.3±1.2 min, respectively; N=33-38 thrombi; p&lt;0.0001). Thrombi size, as determined by platelet fluorescence intensity, were significantly smaller in EC-ATG7 -/- vs. WT mice, (1.8±0.7 and 8.2±2.9, respectively; x10 9 ; p=0.049). Conclusions: Knockdown of ATG7 resulted in reduced TF expression in vitro and in vivo. Compared to WT mice, EC-ATG7 -/- mice exhibited 2-fold greater occlusion times and reduced thrombi formation across all models of thrombosis. Taken together, these findings are evidence that endothelial autophagy regulates thrombosis in a TF-dependent manner, and underscores a novel and previously unrecognized target for antithrombotic drug development.

ENDOTHELIAL AUTOPHAGY REGULATES ARTERIAL THROMBUS FORMATION IN MICE

Arterial thrombosis occurs after rupture of an atherosclerotic plaque, wherein endothelial cells (ECs) become activated and express procoagulant molecules. The role of ECs in thrombosis has not been fully elucidated. Recent evidence suggests that loss of autophagy, an intracellular catabolic process, decreases von Willebrand factor secretion in cultured ECs and in mice. Inasmuch as ATG7 is a key catalyst of autophagy, we explored the potential role of autophagy in thrombogenesis using EC-specific ATG7 knockout (EC-ATG7-/-) mice and their wild-type (WT) littermate controls. Thrombosis was evaluated using intravital microscopy in FeCl3-induced carotid and mesenteric, and laser-induced cremaster artery injury models. Circulating platelet counts were enumerated and platelet surface expression of P-selectin and β3-integrin (β3) determined by fluorescence-activated cell sorting. Time to carotid artery occlusion was significantly prolonged in EC-ATG7-/- mice compared with WT mice (15.5±2.0 and 31.0±4.0 min, respectively; N=10/group; p=0.003). Time to mesenteric artery occlusion was also significantly longer in EC-ATG7-/- mice compared with WT mice (20.0±1.4 and 26.6±1.8 min, respectively; N=10/group; p=0.008). Thrombi in laser injured cremaster arterioles were examined in 38 WT and 33 EC-ATG7-/- mice. The time to maximum platelet fluorescence intensity in EC-ATG7-/- mice was significantly prolonged relative to that for WT mice (1.0±0.6 and 2.3±1.2 min, respectively; p<0.0001). Thrombi size, as determined by platelet fluorescence intensity, were significantly smaller in EC-ATG7-/- vs. WT mice, (1.8±0.7 and 8.2±2.9, respectively; x109; p=0.049). Despite these differences, platelet counts for WT and EC-ATG7-/- mice were not appreciably different (768±86 and 715±125, respectively; x106; N=10; p=0.3). In WT and EC-ATG7-/- mice, surface expression of β3 (68.4±24.4 and 42.8±21.1 AU, respectively; p=0.7) and P-selectin (6.0±1.2 and 5.2±0.8 AU, respectively; p=0.9) were also similar. Although EC-ATG7-/- mice exhibited extended occlusion times and reduced thrombi formation across all three models of thrombosis, platelet count, P-selectin levels and β3 expression were similar to those in WT mice. Taken together, these findings are evidence that endothelial autophagy contributes to thrombosis in a platelet-independent manner.