Endothelial Autophagy Research Articles

ENDOTHELIAL-SPECIFIC LOSS OF AUTOPHAGY PROMOTES ORGAN FIBROSIS

ENDOTHELIAL-SPECIFIC LOSS OF AUTOPHAGY PROMOTES ORGAN FIBROSIS

Endothelial autophagy and Endothelial-to-Mesenchymal Transition (EndoMT) in eEPC treatment of ischemic AKI.

BackgroundAutophagy enables cells to digest endogenous/exogenous waste products, thus potentially prolonging the cellular lifespan. Early endothelial progenitor cells (eEPCs) protect mice from ischemic acute kidney injury (AKI). The mid-term prognosis in AKI critically depends on vascular rarefication and interstitial fibrosis with the latter partly being induced by mesenchymal transdifferentiation of endothelial cells (EndoMT). This study aimed to determine the impact of eEPC preconditioning with different autophagy inducing agents [suberoylanilide hydroxamic acid (SAHA)/temsirolimus] in ischemic AKI.MethodsMale C57/Bl6 N mice were subjected to bilateral renal ischemia (40 min). Animals were injected with either untreated, or SAHA- or temsirolimus-pretreated syngeneic murine eEPCs at the time of reperfusion. Mice were analyzed 48 h and 4 weeks later. In addition, cultured eEPCs were treated with transforming growth factor (TGF)-β ± SAHA, autophagy (perinuclear LC3-II), and stress-induced premature senescence (SIPS—senescence-associated β-galactosidase, SA-β-Gal), and were evaluated 96 h later.ResultsCultured eEPCs showed reduced perinuclear density of LC3-II + vesicles and elevated levels of SA-β-Gal after treatment with TGF-β alone, indicating impaired autophagy and aggravated SIPS. These effects were completely abrogated by SAHA. Systemic administration of either SAHA or tems pretreated eEPCs resulted in elevated intrarenal endothelial p62 at 48 h and 4 weeks, indicating stimulated endothelial autophagy. This effect was most pronounced after injection of SAHA-treated eEPCs. At 4 weeks endothelial expression of mesenchymal alpha-smooth muscle actin (αSMA) was reduced in animals receiving untreated and SAHA-pretreated cells. In addition, SAHA-treated cells reduced fibrosis at week 4. Tems in contrast aggravated EndoMT. Postischemic renal function declined after renal ischemia and remained unaffected in all experimental cell treatment groups.ConclusionIn ischemic AKI, intrarenal endothelial autophagy may be stabilized by systemic administration of pharmacologically preconditioned eEPCs. Early EPCs can reduce postischemic EndoMT and fibrosis in the mid-term. Autophagy induction in eEPCs either increases or decreases the mesenchymal properties of intrarenal endothelial cells, depending on the substance being used. Thus, endothelial autophagy induction in ischemic AKI, mediated by eEPCs is not a renoprotective event per se.

Abstract 4173: Endoglin regulation of Smad2/3 function mediates beclin1 expression and endothelial autophagy during angiogenesis

Abstract Autophagy is the targeted degradation of proteins and organelles critical for homeostasis and cell survival. Transforming growth factor beta (TGF-beta) is known to differentially regulate autophagy under distinct physiological conditions by inducing inhibitory, stimulatory, or a combination of these opposing signaling pathways. Importantly, how TGF-beta signaling controls autophagic responses in the endothelial cell (EC) system during angiogenesis remains unknown. Here we identified endoglin, an EC-specific TGF-beta co-receptor essential for angiogenesis, as a key determinant of autophagy. We found that Smad2/3 are major transcriptional regulators of basal autophagy that specifically target beclin1 (BECN1) gene expression. Selectively blocking ALK5 activation or silencing endogenous Smad2/3 markedly increased BECN1 expression whereas altering ALK1/Smad1/5/8 signaling had no effect. Endoglin potentiates basal autophagy by suppressing ALK5/Smad2/3 activation. Notably, increased beclin1 levels upon Smad2/3 knockdown directly correlated with enhanced autophagy during angiogenesis. Taken together, these results establish endoglin as a critical mediator of autophagy and demonstrate a new cellular mechanism by which Smad2/3 inhibits angiogenesis. Citation Format: Nam Y. Lee, Christopher Pan, Sanjay Kumar. Endoglin regulation of Smad2/3 function mediates beclin1 expression and endothelial autophagy during angiogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4173. doi:10.1158/1538-7445.AM2015-4173

INCRETIN AMELIORATES DIABETIC MICROANGIOPATHY IN HEART BY ACTIVATION OF AUTOPHAGY VIA PKA/AMPK AXIS.

IntroductionThe incretin hormone glucagon‐like peptide‐1 (GLP‐1) restores diabetic myocardial remodeling observed in type 2 diabetic mice (T2DM); however, it remains unclear whether GLP‐1 may modulates angiogenic activity in diabetic heart.HypothesisWe evaluated the impact of GLP‐1 receptor activation on angiogenesis and its link to endothelial autophagy under diabetic condition.MethodsT2DM mice were treated with Ex4 (24 nmole/kg/day for 4 weeks). Cardiac capiary density was measured by CD31 immunohistological staining. Cultured ECs were used for in vitro experiments. Analyses for the changes in autophagy (LC3‐turnover assay and protein levels of p62 and Beclin1), and angiogenesis (tube formation assay and Akt/AMPK/eNOS activity), were evaluated with or without molecular‐targetted knockdown by RNA interference (siRNA).ResultsT2DM exhibited reduced cardiac capillary density in diabetic heart, which was restored by Ex‐4 with increase in myocardial cyclic AMP concentration. GLP‐1 receptor expression in ECs was confirmed by immunoblot and QPCR. Ex‐4 and PKA enhancers (10 μM forskolin and 1 mM 8‐bromo‐cyclic AMP) facilitated angiogenesis and autophagy in ECs. PKA/AMPK/eNOS phosphorylation levels in ECs were enhanced by Ex4, but each Akt activity remains unchanged. PKA inhibitors (H89, RP‐cAMP, and siRNA for PKA) abrogated the increase in AMPK/eNOS phosphorylation induced by Ex‐4. Tube formation assay revealed that Ex4 enhanced in vitro angiogenesis which were abrogated by inhibition of autophagy and AMPK activity.ConclusionsGLP‐1 receptor activation by Ex‐4 restores impaired angioogenic activity in diabetic heart via the PKA/AMPK‐dependent endothelial autophagic activation.

The Essential Autophagy Gene ATG7 Modulates Organ Fibrosis via Regulation of Endothelial-to-Mesenchymal Transition

Pulmonary fibrosis is a progressive disease characterized by fibroblast proliferation and excess deposition of collagen and other extracellular matrix components. Although the origin of fibroblasts is multifactorial, recent data implicate endothelial-to-mesenchymal transition as an important source of fibroblasts. We report herein that loss of the essential autophagy gene ATG7 in endothelial cells (ECs) leads to impaired autophagic flux accompanied by marked changes in EC architecture, loss of endothelial, and gain of mesenchymal markers consistent with endothelial-to-mesenchymal transition. Loss of ATG7 also up-regulates TGFβ signaling and key pro-fibrotic genes in vitro. In vivo, EC-specific ATG7 knock-out mice exhibit a basal reduction in endothelial-specific markers and demonstrate an increased susceptibility to bleomycin-induced pulmonary fibrosis and collagen accumulation. Our findings help define the role of endothelial autophagy as a potential therapeutic target to limit organ fibrosis, a condition for which presently there are no effective available treatments.

Abstract 13502: Glucagon-like Peptide-1 Directly Promotes Angiogenesis via PKA/AMPK-dependent Autophagy in Endothelial Cells

Introduction: We recently reported the impact of glucagon-like peptide-1 (GLP-1) on myocardial remodeling observed in type 2 diabetic mice (T2DM) via cyclic-AMP-dependent activation of autophagy in myocardium; however, it remains unclear whether GLP-1 may modulates capillary formation in heart. Hypothesis/AIM: We thus evaluated the impact of GLP-1 on angiogenesis and its link to endothelial autophagy. Methods: T2DM was treated with Ex4 (24 nmole/kg/day for 4 weeks). Cardiac capillary density was measured by immunohistology. Cultured HUVECs were used for in vitro experiments. Changes in activities of autophagy (LC3-turnover assay and protein levels of p62 and Beclin1), and angiogenesis (tube formation assay and Akt/AMPK/eNOS activity), were evaluated.[[Unable to Display Character: 
]] Role of PKA was assessed by CREB phosphorylation and RNA interference (siRNA for catalytic subunit of PKA). Effect of autophagy was assessed by use of pharmacological inhibitor 3MA and siRNA of autophagy-related gene (ATG) 5 , ATG7, and p62. Results: Immunohistochemical analyses revealed that T2DM exhibited reduced cardiac capillary density, which was reversed by Ex-4 with concomitant amelioration of systemic diabetic condition. Ex-4 treated heart exhibited increase in myocardial cyclic AMP concentration. We thus observed direct impact of Ex-4 and cyclic AMP elevation on HUVECs, in which GLP-1 receptor expression was confirmed by immunoblot and QPCR. In vitro angiogenesis assay revealed that Ex-4 and PKA enhancers (10 μM forskolin and 1 mM 8-bromo-cyclic AMP) facilitated angiogenesis and autophagy in HUVECs. The PKA/AMPK/eNOS phosphorylation levels of Ex-4-treated HUVECs were elevated. Of note, each Akt activity remains unchanged. PKA inhibitors (H89, RP-cAMP, siRNA) abrogated the increase in phosphorylation of AMPK/eNOS axis induced by Ex-4 in HUVECs. Tube formation assay revealed that Ex-4 and PKA enhancers augmented in vitro angiogenesis, which were abrogated by inhibition of autophagy and AMPK using pharmacological inhibitors (3MA and compound C) and siRNA for autophagy-related gene (ATG) 5 , ATG7, p62, and catalytic subunit of AMPK. [[Unable to Display Character: 
]] Conclusions: GLP-1 directly promoted angiogenesis via the PKA/AMPK-dependent autophagic activation.

ARG2 impairs endothelial autophagy through regulation of MTOR and PRKAA/AMPK signaling in advanced atherosclerosis

Impaired autophagy function and enhanced ARG2 (arginase 2)-MTOR (mechanistic target of rapamycin) crosstalk are implicated in vascular aging and atherosclerosis. We are interested in the role of ARG2 and the potential underlying mechanism(s) in modulation of endothelial autophagy. Using human nonsenescent “young” and replicative senescent endothelial cells as well as Apolipoprotein E-deficient (apoe−/−Arg2+/+) and Arg2-deficient apoe−/− (apoe−/−arg2−/−) mice fed a high-fat diet for 10 wk as the atherosclerotic animal model, we show here that overexpression of ARG2 in the young cells suppresses endothelial autophagy with concomitant enhanced expression of RICTOR, the essential component of the MTORC2 complex, leading to activation of the AKT-MTORC1-RPS6KB1/S6K1 (ribosomal protein S6 kinase, 70kDa, polypeptide 1) cascade and inhibition of PRKAA/AMPK (protein kinase, AMP-activated, α catalytic subunit). Expression of an inactive ARG2 mutant (H160F) had the same effect. Moreover, silencing RPS6KB1 or expression of a constitutively active PRKAA prevented autophagy suppression by ARG2 or H160F. In senescent cells, enhanced ARG2-RICTOR-AKT-MTORC1-RPS6KB1 and decreased PRKAA signaling and autophagy were observed, which was reversed by silencing ARG2 but not by arginase inhibitors. In line with the above observations, genetic ablation of Arg2 in apoe−/− mice reduced RPS6KB1, enhanced PRKAA signaling and endothelial autophagy in aortas, which was associated with reduced atherosclerosis lesion formation. Taken together, the results demonstrate that ARG2 impairs endothelial autophagy independently of the L-arginine ureahydrolase activity through activation of RPS6KB1 and inhibition of PRKAA, which is implicated in atherogenesis.

ENDOTHELIAL AUTOPHAGY REGULATES CHOLESTEROL TRANSPORT AND IS A NOVEL REGULATOR OF ATHEROSCLEROSIS

nectin inhibited these outcomes. Compared to monocytes from Adipoq+/+ mice, those from Adipoq-/littermates showed a w6-fold greater production of MPs (Cd11b+/ Ly6Chi/AnnexinV+) following LPS stimulation. CONCLUSION: We report a novel mechanism through which mono-MPs sustain NLRP3 inflammasome activation and endothelial dysfunction. These data reveal a previously unknown atheroprotective mechanism for adiponectin that may account for the inverse association observed between circulating adiponectin levels and the risk of diabetes and obesity.

EEOC-mediated modulation of endothelial autophagy, senescence, and EnMT in murine diabetic nephropathy.

Diabetic nephropathy is the most frequent single cause of end-stage renal disease in our society. Microvascular damage is a key event in diabetes-associated organ malfunction. Early endothelial outgrowth cells (eEOCs) act protective in murine acute kidney injury. The aim of the present study was to analyze consequences of eEOC treatment of murine diabetic nephropathy with special attention on endothelial-to-mesenchymal transdifferentiation, autophagy, senescence, and apoptosis. Male C57/Bl6N mice (8-12 wk old) were treated with streptozotocin for 5 consecutive days. Animals were injected with untreated or bone morphogenetic protein (BMP)-5-pretreated syngeneic murine eEOCs on days 2 and 5 after the last streptozotocin administration. Four, eight, and twelve weeks later, animals were analyzed for renal function, proteinuria, interstitial fibrosis, endothelial-to-mesenchymal transition, endothelial autophagy, and senescence. In addition, cultured mature murine endothelial cells were investigated for autophagy, senescence, and apoptosis in the presence of glycated collagen. Diabetes-associated renal dysfunction (4 and 8 wk) and proteinuria (8 wk) were partly preserved by systemic cell treatment. At 8 wk, antiproteinuric effects were even more pronounced after the injection of BMP-5-pretreated cells. The latter also decreased mesenchymal transdifferentiation of the endothelium. At 8 wk, intrarenal endothelial autophagy (BMP-5-treated cells) and senescence (native and BMP-5-treated cells) were reduced. Autophagy and senescence in/of cultured mature endothelial cells were dramatically reduced by eEOC supernatant (native and BMP-5). Endothelial apoptosis decreased after incubation with eEOC medium (native and BMP-5). eEOCs act protective in diabetic nephropathy, and such effects are significantly stimulated by BMP-5. The cells modulate endothelial senescence, autophagy, and apoptosis in a protective manner. Thus, the renal endothelium could serve as a therapeutic target in diabetes-associated kidney dysfunction.

Hyperphosphatemia induces protective autophagy in endothelial cells through the inhibition of Akt/mTOR signaling

Hyperphosphatemia-induced endothelial dysfunction has been shown to play a pathogenic role in the development of atherosclerosis in chronic kidney disease (CKD) through unclear mechanisms. Emerging evidence indicates that autophagy is involved in the maintenance of normal cardiovascular function. However, it is unclear whether autophagy participates in the molecular mechanism underlying high phosphate (Pi)-induced endothelial dysfunction. The autophagy activity was determined by the immunofluorescence staining of the expression of endothelial microtubule-associated protein 1 light chain 3 (LC3) in the 5/6 nephrectomy rat model of CKD and sham-operated control rats. The LC3-II/LC3-I ratio and the activation of the Akt/mammalian target of rapamycin (mTOR) signaling pathway were determined in cultured human microvascular endothelial cell (HMEC-1) endothelial cells that were exposed to a high concentration of Pi with or without the Pi influx blocker phosphonoformic acid, the autophagy inhibitor 3-methyladenine, and the autophagy inducer rapamycin. The impacts of autophagy on Pi-induced apoptotic damage were assessed by flow cytometry. The in vivo rat model of CKD revealed that hyperphosphatemia is associated with increased endothelial LC3 staining. The exposure of HMEC-1 cells to high Pi induced both dose-dependent and time-dependent increases in the LC3-II/LC3-I expression ratio accompanied by the inhibition of the Akt/mTOR signaling pathway. In HMEC-1 cells, high Pi-induced autophagy and the inhibition of Akt/mTOR signaling were reversed by phosphonoformic acid through the blockage of Pi influx. Apoptosis, characterized by the levels of cleaved caspase 3 and poly(ADP-ribose) polymerase, along with autophagy was induced by high Pi, and the inhibition of autophagy by 3-methyladenine significantly aggravated high Pi-induced apoptosis. The flow cytometry results confirmed that the blockage of autophagy promoted the apoptosis of endothelial cells. Hyperphosphatemia induces endothelial autophagy, possibly through the inhibition of the Akt/mTOR signaling pathway, which may play a protective role against high Pi-induced apoptosis.

Abstract 18846: The Dual Effects of Shear Stress on Vascular Endothelial Autophagy and Mitochondrial DNA Damage

Vascular oxidative stress preferentially develops at arterial curvatures and branching points where oscillatory shear stress (OSS) occurs. Autophagy, a tightly regulated intracellular bulk degradation/recycling system, is implicated in endothelial homeostasis. In this study, we assessed the effects of OSS on autophagy and mitochondrial DNA (mtDNA) damage. Human aortic endothelial cells (HAEC) exposed to OSS (τ av g = 0±3 dyne/cm 2 for 4 hours) increased relative LC3-II/LC3-I ratios (control: 1.0±0.02, OSS: 2.4±0.51, p <0.01, n=4), as supported by an increase in autophagosome punctures by LC3-green fluorescence protein (LC3-GFP). OSS-induced increase in LC3-II/LC3-I ratios were significantly attenuated in response to N-acetyl cysteine (NAC), MnSOD over-expression, and JNK siRNA (siJNK) knockdown ( p <0.05, n=4). Inhibition of autophagy by ATG5 siRNA increased OSS-induced MitoSox intensity for mitochondrial superoxide (mtO 2 •- ) by 2.03±0.25-fold ( p < 0.05, n=4), whereas Rapamycin-induced autophagy reduced OSS induced MitoSox intensity by 78.4% ( p <0.05, n=4). OSS-induced reduction of mitochondrial complex II activity was also attenuated in response to Rapamycin (OSS: 62.1±11.2%; Rapamycin+OSS: 130.1±40.4% (relative to static control), p <0.05, n=4). In comparison with static and pulsatile flow (PSS=23 dyne/cm 2 ) conditions, OSS also significantly increased mtDNA damage by 4.5±1.3-fold ( p <0.01, n=4), which was significantly reversed in response to NAC, MnSOD over-expression, and siJNK knockdown ( p <0.05, n=4). In the fat-fed New Zealand White rabbits, quantification for immuno-staining revealed prominent endothelial JNK phosphorylation and DNA damage in the OSS-exposed aortic arch in contrast with nearly absent staining in the PSS-exposed descending aorta ( p < 0.05, n=4). Furthermore, analysis of caspase-3 activity was negative for OSS-exposed HAEC and OSS-exposed aorta area from normal diet-fed animals. Despite atherogenic nature of OSS, our findings suggest that OSS imparts dual effects in endothelial autophagy and mtDNA damage with an implication in maintaining homeostasis.

Abstract WP442: Oxidative Stress And Cyclic Strain Mediated Smad6 Regulation In Vascular Endothelial Cells Correlates With Autophagy Induction And Endothelial Dysfunction

Background: Vascular endothelial cells (ECs) are subjected to enhanced-oxidative stress and -cyclic strain in hypertension, stroke and atherosclerotic vascular disease. The role for TGF-ß and bone morphogenic proteins (BMPs) has been implied in vascular development and vessel remodeling. Although endothelial specific regulation of their intracellular signaling protein Smad6 was observed under the stimuli of laminar shear stress, little is known about the function of Smad6 in adult vascular endothelium. Methods: Human aortic ECs were treated with H202 (10 to 100umol/L) or exposed to 7% uniaxial cyclic strain. For transient knock down, ECs were transfected with siRNA constructs against Smad6 and Smad7. Expression levels of Smad6 and Smad7 were measured by quantitative RT-PCR and western blotting analysis. NO production and BrdU labeling for proliferative cells were measured by ELISA assay. In vitro angiogenesis was evaluated in tube-formed ECs. The process of autophagy was analyzed by electron microscopy. Cells in survival or apoptosis were detected by immunofluorescence cell staining of HMGB1 or cleaved- caspase-3. Results: Exposure of ECs to 24 hours of oxidative stress or cyclic strain showed consistent reduction in Smad6 mRNA and protein, while no change in Smad7. Notably, electron microscopic analysis showed that knockdown of endogenous Smad6 expression induced “self-eating” autophagy after 24 hours of treatment, while silencing of Smad7 did not induce autophagy. Immunofluorescence cell staining showed that EC with silencing Smad6 were positive for HMGB1 but negative for cleaved caspase-3. Importantly, ECs with silencing Smad6 represented significant reduction in NO production, prolifereative activities and in vitro angiogenesis as compared to those with silencing Smad7. Furthermore, treatment with TGF-ß1, BMP-2, or BMP-4 did not show synergistic effects on autophagy induction and endothelial function after silencing Smad6. Conclusions: These data demonstrate a novel mechanism for functional regulation of Smad6 in vascular ECs, and indicate that dysregulation of Smad6 may have important implications in endothelial autophagy and endothelial dysfunction in the settings of hypertension, stroke and vascular inflammation.

Abstract 521: Role of Autophagy in Endothelial Cells for Atherosclerosis

Endothelial dysfunction is thought to play an important role for the development of atherosclerosis. Autophagy is the mechanism by which organelles, aggregated protein, and even lipid droplet are broken down. Since hyperlipidemia can cause endothelial cell dysfunction during the early stage of atherosclerosis, there may be link between atherosclerosis and endothelial autophagy. To address this issue, we generated endothelial specific Atg7 knockout in apoE deficient mice (Atg7 endo /apoE). En face analysis of Oil-red O stained whole aorta showed Atg7 endo /apoE double knockout mice had severe atherosclerosis. At 16 week old, apoE single knockout mice (apoE) had 9.7 % of oil-red O positive area while Atg7 endo /apoE mice had 18.6 % (n=10, 9 mice per group). The composition of macrophages, collagen, and lipid in atherosclerotic lesions were similar between control and Atg7 endo /apoE mice. Body weight and fat composition were also similar between the two groups, as were blood glucose, triglyceride, cholesterol, and fatty acid levels. At 24 weeks of age, en face analysis of aorta showed even more disparity in lesion size between control and Atg7 endo /apoE mice (apoE mice, 15.4%, Atg7 endo /apoE mice, 36.2%, n=10, 7 mice per group). Secretion of various chemokine, such as MCP-1 from Atg7 knockdown human umbilical vein endothelial cell (shATG7 HUVECs) was 10-fold higher that of control cells. These results suggest that autophagy deficiency in endothelial cells accelerate in vivo atherosclerosis. This augmentation may result from an enhanced inflammatory response.

Abstract 257: Autophagy in Endothelial Cell Has an Important Role for Hemostasis and Thrombosis

Autophagy has been reported to maintain intracellular homeostasis through lysosomal-mediated degradation of damaged organelles and proteins. Recent evidence suggests that autophagy regulates many physiological and pathological process including aging, reactive oxygen species levels, and inflammation. The role of autophagy in endothelial cell biology is poorly understood. Electron microscopic analysis of human umbilical vein endothelial cell (HUVEC) surprisingly demonstrated spontaneous fusion of autophagosomes with Weibel-Palade bodies, endothelial specific organelles containing abundant von Willebrand factor (VWF). Knockdown of the essential autophagy gene Atg7 resulted in a reduction in release of VWF from HUVECs after histamine (2.07±0.02 mU/mL vs. 1.30±0.02 mU/mL), or vascular endothelial growth factor (3.10±0.01 mU/mL vs. 1.22±0.03 mU/mL) stimulation. Pharmacological inhibition of autophagy showed similar results. We generated mice that have an endothelial specific deletion of Atg7. These mice have normal blood pressure (Control; 113±8/87±6 mmHg vs. Atg7 deficient; 119±7/88±4 mmHg) and normal vessel architecture. However, endothelial specific Atg7 deficient mice showed a reduction of plasma VWF and marked elevation of their bleeding times (The number of mice with bleeding time of more than 10min; Control=1/17, Atg7 deficient 6/9). Thus, an intact endothelial autophagy machinery is necessary for VWF secretion and hemostasis.

The Factors Influence Vascular Endothelial Cells of Autophagy and Related Mechanism*

Autophagy plays critical role in maintaining the cell homeostasis,organelle turnover and nutrient recycling in physiological condition.Autophagy is consist in the body's physiological and pathological process.It has protection and repairing role at basic autophagy level,but over-autophagy would lead to injury and apoptosis.In the past years,major studies are focussing on cancer cell autophagy,and only a few studies concerning about normal cells.Vascular endothelial cells as one of the most active cells in the human body,many cardioascular disease were bound up with vascular endothelial cells(VECs) function change.This review summarizes influence factors on vascular endothelial cells autophagy.

Distinct patterns of autophagy evoked by two benzoxazine derivatives in vascular endothelial cells

Macroautophagy (referred to as autophagy) is an evolutionarily conserved, bulk-destruction process in eukaryotes. During this process, the cytoplasm containing long-lived proteins and organelles is engulfed into double-membrane autophagosomes, and ultimately undergoes enzymatic degradation within lysosomes. Autophagy serves as a prosurvival machinery, or it may contribute to cell death. Accumulating evidence indicates that autophagy is involved in the pathogenesis and intervention of various human diseases. Pharmacological autophagy modulators are arousing interest from biologists and clinical physicians in light of their potential for disease therapy and increasing our understanding of the mechanism of autophagy. In this study, we identified two autophagy enhancers, 6-amino-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine (ABO) and 6,8-dichloro-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine (DBO), in human umbilical vein endothelial cells (HUVEC s) by autophagy assays, and demonstrate that ABO and DBO could stimulate autophagy in an mtor-independent and mtor-dependent manner, respectively; ABO-stimulated autophagy was attributed to the elevation of the Ca2+ channel annexin A7 (ANXA7), whereas DBO’s effect was due to the level of intracellular reactive oxygen species (ROS). Importantly, we found that ANXA7 was essential for autophagy induction via modulating the intracellular calcium concentration ([Ca2+]i) in HUVEC s. In summary, our work introduced two distinct autophagy enhancers and highlighted the critical role of ANXA7 in endothelial autophagy.

Alzheimer Disease–Associated Peptide, Amyloid β40, Inhibits Vascular Regeneration With Induction of Endothelial Autophagy

Although the majority of cases of Alzheimer disease (AD) are known to be attributable to the sporadic (nongenetic) form of the disease, the mechanism underlying its cause and progression still remains unclear. We found that vascular beta-amyloid (Abeta), Abeta40, inhibited the proliferative activity of human brain vascular endothelial cells (HBECs) without toxic effects on them. This peptide also inhibited tube formation and migration of HBECs. Moreover, Abeta40 inhibited ex vivo hippocampal revascularization, reendothelialization, and the differentiation of adult endothelial progenitor cells. Importantly, Abeta40 suppressed the proliferative activity of HBECs through the induction of "self-digesting" autophagy. This induction involved the intracellular regulation of class 3 phosphatidylinositol 3-kinase (PI3K) as well as Akt signaling in HBECs. Furthermore, tissue culture of murine brain sections from GFP-LC3 transgenic mice revealed that Abeta40 not only reduced the vessel density in hippocampal lesions, but also induced autophagy in neurovascular ECs. Our present findings indicate that the initial progression of AD might be in part driven by Abeta40-induced endothelial autophagy and impairment of neurovascular regeneration, suggesting important implications for therapeutic approaches to AD.