Autophagy In Vascular Endothelial Cells Research Articles

The role and mechanism of action of miR‑92a in endothelial cell autophagy.

Although microRNAs (miRNAs/miRs) serve a significant role in the autophagy of vascular endothelial cells (ECs), the effect of miR‑92a on the autophagy of ECs is currently unclear. Therefore, the present study aimed to investigate the impact of miR‑92a on autophagy in ECs and the underlying molecular processes that control this biological activity. Firstly, an autophagy model of EA.hy926 cells was generated via treatment with the autophagy inducer rapamycin (rapa‑EA.hy926 cells). The expression levels of miR‑92a were then detected by reverse transcription‑quantitative PCR, and the effect of miR‑92a expression on the autophagic activity of rapa‑EA.hy926 cells was studied by overexpressing or inhibiting miR‑92a. The level of autophagy was evaluated by western blot analysis, immunofluorescence staining and transmission electron microscopy. Dual‑luciferase reporter assays were used to confirm the interaction between miR‑92a and FOXO3. The results demonstrated that the expression levels of miR‑92a were decreased in the rapa‑EA.hy926 cell autophagy model. Furthermore, overexpression and inhibition of miR‑92a revealed that upregulation of miR‑92a in these cells inhibited autophagy, whereas miR‑92a knockdown promoted it. It was also confirmed that miR‑92a directly bound to the 3'‑untranslated region of the autophagy‑related gene FOXO3 and reduced its expression. In conclusion, the present study suggested that miR‑92a inhibits autophagy activity in EA.hy926 cells by targeting FOXO3.

The Role of Autophagy in Vascular Endothelial Cell Health and Physiology.

Autophagy is a highly conserved cellular recycling process which enables eukaryotes to maintain both cellular and overall homeostasis through the catabolic breakdown of intracellular components or the selective degradation of damaged organelles. In recent years, the importance of autophagy in vascular endothelial cells (ECs) has been increasingly recognized, and numerous studies have linked the dysregulation of autophagy to the development of endothelial dysfunction and vascular disease. Here, we provide an overview of the molecular mechanisms underlying autophagy in ECs and our current understanding of the roles of autophagy in vascular biology and review the implications of dysregulated autophagy for vascular disease. Finally, we summarize the current state of the research on compounds to modulate autophagy in ECs and identify challenges for their translation into clinical use.

PCSK9 induces endothelial cell autophagy by regulating the PI3K/ATK pathway in atherosclerotic coronary heart disease.

Atherosclerosis is a chronic inflammatory disease of the arteries, and its pathogenesis is related to endothelial dysfunction. It has been found that the protein convertase subtilin/kexin9 type (PCSK9) plays an important role in AS, but its specific mechanism is still unclear. In this study, we first cultured human umbilical vein endothelial cells (HUVECs) with 50 or 100μg/ml oxidized low-density lipoprotein (ox-LDL) for 24 hours to establish a coronary atherosclerosis cell model. The results showed that ox-LDL induced HUVEC injury and autophagy and upregulated PCSK9 protein expression in HUVECs in a concentration-dependent manner. Silencing PCSK9 expression with siRNA inhibited ox-LDL-induced HUVEC endothelial dysfunction, inhibited the release of inflammatory factors, promoted HUVEC proliferation and inhibited apoptosis. In addition, ox-LDL increased the expression of LC3B-I and LC3B-II and decreased the expression of p62. However, these processes are reversed by sh-PCSK9. In addition, sh-PCSK9 can inhibit PI3K, AKT and mTOR phosphorylation and promote autophagy. Taken together, our research shows that silencing PCSK9 inhibits the PI3K/ATK/mTOR pathway to activate ox-LDL-induced autophagy in vascular endothelial cells, alleviating endothelial cell injury and inflammation.

NBR1-mediated autophagic degradation of caspase 8 protects vascular endothelial cells against arsenite-induced apoptotic cell death

Vascular endothelial cells play a critical role in maintaining the health of blood vessels, but dysfunction can lead to cardiovascular diseases. The impact of arsenite exposure on cardiovascular health is a significant concern due to its potential adverse effects. This study aims to explore how NBR1-mediated autophagy in vascular endothelial cells can protect against oxidative stress and apoptosis induced by arsenite. Initially, our observations revealed that arsenite exposure increased oxidative stress and triggered apoptotic cell death in human umbilical vein endothelial cells (HUVECs). However, treatment with the apoptosis inhibitor Z-VAD-FMK notably reduced arsenite-induced apoptosis. Additionally, arsenite activated the autophagy pathway and enhanced autophagic flux in HUVECs. Interestingly, inhibition of autophagy exacerbated arsenite-induced apoptotic cell death. Our findings also demonstrated the importance of autophagy receptor NBR1 in arsenite-induced cytotoxicity, as it facilitated the recruitment of caspase 8 to autophagosomes for degradation. The protective effect of NBR1 against arsenite-induced apoptosis was compromised when autophagy was inhibited using pharmacological inhibitors or through genetic knockdown of essential autophagy genes. Conversely, overexpression of NBR1 facilitated caspase 8 degradation and reduced apoptotic cell death in arsenite-treated HUVECs. In conclusion, our study highlights the vital role of NBR1-mediated autophagic degradation of caspase 8 in safeguarding vascular endothelial cells from arsenite-induced oxidative stress and apoptotic cell death. Targeting this pathway could offer a promising therapeutic approach to mitigate cardiovascular diseases associated with arsenite exposure.

Proteoglycans of basement membranes: Crucial controllers of angiogenesis, neurogenesis, and autophagy.

Anti-angiogenic therapy is an established method for the treatment of several cancers and vascular-related diseases. Most of the agents employed target the vascular endothelial growth factor A, the major cytokine stimulating angiogenesis. However, the efficacy of these treatments is limited by the onset of drug resistance. Therefore, it is of fundamental importance to better understand the mechanisms that regulate angiogenesis and the microenvironmental cues that play significant role and influence patient treatment and outcome. In this context, here we review the importance of the three basement membrane heparan sulfate proteoglycans (HSPGs), namely perlecan, agrin and collagen XVIII. These HSPGs are abundantly expressed in the vasculature and, due to their complex molecular architecture, they interact with multiple endothelial cell receptors, deeply affecting their function. Under normal conditions, these proteoglycans exert pro-angiogenic functions. However, in pathological conditions such as cancer and inflammation, extracellular matrix remodeling leads to the degradation of these large precursor molecules and the liberation of bioactive processed fragments displaying potent angiostatic activity. These unexpected functions have been demonstrated for the C-terminal fragments of perlecan and collagen XVIII, endorepellin and endostatin. These bioactive fragments can also induce autophagy in vascular endothelial cells which contributes to angiostasis. Overall, basement membrane proteoglycans deeply affect angiogenesis counterbalancing pro-angiogenic signals during tumor progression, and represent possible means to develop new prognostic biomarkers and novel therapeutic approaches for the treatment of solid tumors.

Arctigenin mitigates vascular endothelial injury in rats with pregnancy-induced hypertension via autophagy-NLRP3 inflammasome pathway

This study aims to investigate the effect and mechanism of arctigenin(ARC) in the treatment of vascular endothelial injury in rats with pregnancy-induced hypertension(PIH). Fifty SD rats pregnant for 12 days were randomly assigned into a control group, a model group, an ARC group, a rapamycin(RAP, autophagy inducer) group, and an ARC+3-methyladenine(3-MA, autophagy inhibitor) group, with 10 rats in each group. The rats in the other groups except the control group were intraperitoneally injected with nitrosyl-L-arginine methyl ester(50 mg·kg~(-1)·d~(-1)) to establish the PIH model on the 13th day of pregnancy. On the 15th day of pregnancy, the rats in ARC, RAP, and ARC+3-MA groups were intraperitoneally injected with ARC(50 mg·kg~(-1)·d~(-1)), RAP(1 mg·kg~(-1)·d~(-1)), and 3-MA(15 mg·kg~(-1)·d~(-1))+ARC(50 mg·kg~(-1)·d~(-1)), respectively. The pregnant rats in the control group and the model group were intraperitoneally injected with the same amount of normal saline. The blood pressure and 24 h urine protein(24 h-UP) of pregnant rats in each group were measured before and after intervention. Cesarean section was performed to terminate pregnancy on day 21, and the body weight and body length of fetal rats were compared among groups. Hematoxylin-eosin(HE) staining was employed to observe the pathological changes of placenta. The expression of endothelin-1(ET-1) and endothelial nitric oxide synthase(eNOS) in placenta was detected by immunohistochemistry. The serum levels of ET-1 and nitric oxide(NO) were determined with corresponding kits. The expression of microtubule-associated protein 1 light chain 3(LC3), Beclin-1, NOD-like receptor protein 3(NLRP3), apoptosis-associated speck-like protein with CARD domain(ASC), caspase-1, interleukin(IL)-1β, and IL-18 was determined by immunofluorescence and Western blot. The level of reactive oxygen species(ROS) in placenta was measured by fluorescence staining. The results showed that on day 12 of pregnancy, the blood pressure and 24 h-UP had no significant differences among groups. On days 15, 19, and 21, the blood pressure and 24 h-UP in the model group were higher than those in the control group(P<0.05). On days 19 and 21, the blood pressure and 24 h-UP in ARC group and RAP group were lower than those in the model group(P<0.05), and they were higher in the ARC+3-MA group than in the ARC group(P<0.05). On day 21, the model group had lower body weight and body length of fetal rats(P<0.05), higher serum level of ET-1, and lower serum level of NO(P<0.05) than the control group. Moreover, the placental tissue showed typical pathological damage, down-regulated expression of LC3-Ⅱ/LC3-Ⅰ, Beclin-1 and eNOS(P<0.05), up-regulated expression of ET-1, NLRP3, ASC, caspase-1, IL-1β, and IL-18(P<0.05), and elevated ROS level. Compared with the model group, ARC and RAP groups showed increased body weight and body length of fetal rats(P<0.05), lowered serum level of ET-1, elevated serum level of NO(P<0.05), reduced pathological damage of placental tissue, up-regulated expression of LC3-Ⅱ/LC3-Ⅰ, Beclin-1, and eNOS(P<0.05), down-regulated expression of ET-1, NLRP3, ASC, caspase-1, IL-1β, and IL-18(P<0.05), and lowered ROS level. Compared with ARC group, 3-MA reversed the effects of ARC on the above indicators. In conclusion, ARC can inhibit the activation of NLRP3 inflammasome and mitigate vascular endothelial damage in PIH rats by inducing autophagy of vascular endothelial cells.

Dl-3-n-butylphthalide improves intestinal microcirculation disorders in septic rats by regulating the PI3K/AKT signaling pathway and autophagy.

Sepsis has complex pathophysiological mechanisms that bring new challenges in the treatment of sepsis at a time when the intestinal microcirculation in sepsis is receiving increasing attention. Dl-3-n-butylphthalide (NBP), which is a drug that can improve multiorgan ischemic diseases, is also worth examining to improve the intestinal microcirculation in sepsis. In this study, male Sprague-Dawley rats were divided into the sham group (n=6), CLP group (n=6), NBP group (n=6) and NBP+LY294002 group (n=6). The rat model of severe sepsis was established by cecal ligation and puncture (CLP). Abdominal wall incisions and sutures were performed in the first group, and CLP was performed in the latter three groups. Normal saline/NBP/NBP+LY294002 solution was injected intraperitoneally 2h or 1h before modeling. Hemodynamic data (blood pressure and heart rate) were recorded at 0, 2, 4 and 6h. Sidestream dark field (SDF) imaging and the Medsoft System were used to observe the intestinal microcirculation of rats and obtain data at 0, 2, 4, and 6h. Six hours after the model was established, the serum levels of TNF-α and IL-6 were measured to evaluate the level of systemic inflammation. Pathological damage to the small intestine was evaluated by electron microscopy and histological analysis. The expression levels of P-PI3K, PI3K, P-AKT, AKT, LC3 and p62 in the small intestine were analyzed by Western blotting. The expressions of P-PI3K, P-AKT, LC3 and P62 in small intestine were detected by immunohistochemical staining. NBP improved intestinal microcirculation disturbances in septic rats, alleviated the systemic inflammatory response, reduced the destruction of the small intestinal mucosa and the disruption of microvascular endothelial cells, and alleviated autophagy in vascular endothelial cells. NBP increased the ratio of P-PI3K/total PI3K, P-AKT/total AKT, and P62/β-actin and decreased the ratio of LC3 II/LC3 I. NBP ameliorated intestinal microcirculation disturbances and the destruction of small intestinal vascular endothelial cells in septic rats by activating the PI3K/Akt signaling pathway and regulating autophagy.

LncRNA TGFB2-OT1 Promotes Progression and Angiogenesis in Hepatocellular Carcinoma by Dephosphorylating β-Catenin.

Hepatocellular carcinoma (HCC) was the sixth most prevalent cancer worldwide. Long non-coding RNA TGFB2-OT1 has been proven to mediate inflammation and autophagy in vascular endothelial cells. However, its function in HCC is still unknown. We analyzed the relationship between TGFB2-OT1 expression and the clinicopathological features of 202 HCC patients. RT-qPCR was used to analyze the TGFB2-OT1 expression in HCC cell lines and tissues. In vitro and in vivo assays were conducted to verify the effect of TGFB2-OT1 on the phenotype of HCC. RNA pull-down assays were applied to reveal the proteins binding to the TGFB2-OT1. Western-blot assays were conducted to analyze the protein expression in HCC cell lines. TGFB2-OT1 was found to be highly expressed in HCC samples and hepatoma cells. TGFB2-OT1 expression was significantly associated with age (P = 0.001), cirrhosis (P = 0.003), tumor size (P < 0.001), tumor encapsulation (P = 0.029), tumor protruding from the liver surface (P = 0.040), and alpha fetoprotein (AFP, P < 0.001) levels. TGFB2-OT1 promoted proliferation, migration, invasion, and angiogenesis in HCC cells, both in vitro and in vivo. TGFB2-OT1 binds to β-catenin and competitively impaired the binding of β-catenin to GSK3β, thus suppressing the phosphorylation of β-catenin at Ser33, Ser37, and Thr41. TGFB2-OT1 is overexpressed in HCC and predicts the poor prognosis of HCC patients. TGFB2-OT1 impedes the phosphorylation of β-catenin and acts as an alternative activator of the Wnt/β-catenin pathway to promote the progression and angiogenesis of HCC.

Protective effects of salidroside on vascular endothelial cells in rats with frostbite after chronic hypoxia

Objective: To investigate the protective effects of salidroside on endothelial cells in rats with frostbite after chronic hypoxia. Methods: Healthy male SD rats, randomly divided into 3 groups with 10 rats in each group, which included the sham injury group, the model group, and the model +salidroside group. The rats in each group were placed in a composite low-pressure chamber to simulate a environment with a pressure of 54.1 kpa and a temperature of 23~25°C. The rats were exposed to hypoxia under these conditions for 14 days, during the experimental time the rats in the model+salidroside group were treated with 50 mg/kg salidroside daily. After the rats were removed from the low-pressure chamber, except for the sham injury group, frozen iron sheets were applied tightly to the back of the rats for 30 s, supplemented with low temperature for frostbite modeling. Blood and skin tissues were collected at 12 hours after modeling for testing. The structural changes in tissue and vascular endothelial cells were observed in the frostbite region. Vascular endothelial cell particulate EMP levels were detected. The levels of ICAM-1, sEPCR, vWF, ET-1 and NO secretion were determined. The expression levels of HIF-1α, p-PI3K, p-Akt and VEGF were detected by Western blot. Results: Salidroside could effectively reduce skin collapse in frostbitten areas. It could reduce the injury of frostbitten tissues, and improve the subcutaneous tissue necrosis and inflammatory cell infiltration. The autophagy of vascular endothelial cells was reduced. Compared with the model group (0.250±0.165)%, the expression of EMPs in the model+salidroside group (2.453±0.196)% was increased significantly (P＜0.01). In addition, the contents of NO (2.622±0.219)pg/ml was also significantly higher than that of the model group (1.616±0.152)pg/ml (P＜0.01), and the content of vWF (233.50±13.43)pg/ml was lower than that of the model group (315.60±8.78)pg/ml (P＜0.05). There was no significant difference in the levels of ICAM-1, sEPCR and ET-1. Salidroside significantly decreased the expressions of p-PI3K, p-Akt, VEGF and HIF-1α protein in vascular endothelial cells of rats with frostbite (P＜0.01). Conclusion: Salidroside can reduce endothelial cell damage, reduce endothelial cell autophagy and promote endothelial cell regeneration. Based on the PI3K/Akt pathway, salidroside has a good protective effect on endothelial cells of rats with frostbite after chronic hypoxia.

Nucleus-Targeted Nanoparticles Induce Autophagy of Vascular Endothelial Cells in Cervical Spondylosis of Vertebral Artery Type Through PI3K/Akt/mTOR Signaling Pathway.

Nanoparticles are characterized by their large surface area per unit and high dispersion, with excellent affinity and adhesion to the tissue, which help them to contact drugs with tissues. However, the relationship between nuclear-targeted nanoparticles and PI3K/Akt/mTOR pathway, as well as their roles in cervical spondylosis of vertebral artery type (CSA) remain unclear. bEnd.3 cells were in this study exposed to nuclear-targeted nanoparticles, followed by determination of cell biological processes. The role of nuclear-targeted nanoparticles in CSA in relation to PI3K/Akt/mTOR pathway was then analyzed through detection of autophagy-related proteins pathway-related proteins. Nuclear-targeted nanoparticles led to reduced bEnd.3 cell proliferation with IC50 at indicated time points shown as (12.8±0.67), (8.8±0.43), and (4.6±0.42) μmol/L, respectively. Nuclear-targeted nanoparticles blocked bEnd.3 cells in G2/M phase, and induced apoptosis. In addition, nuclear-targeted nanoparticles inhibited the PI3K/Akt/mTOR pathway in the bEnd.3 cells, as evidenced by reduced PI3K, Akt and mTOR levels. Nuclear-targeted nanoparticles decreased the expression of Beclin-1, LC3, p62, Cathepsin D, and ATG5, and increased expression of GSK-3 and Bcl-2. Our present study demonstrated that the nucleartargeted nanoparticles could regulate the growth of bEnd.3 cells in CSA and promote autophagy of cells through blockage of the PI3K/Akt/mTOR signaling pathway.

Autophagy and skin wound healing.

Autophagy is a lysosome-dependent, self-renewal mechanism that can degrade and recycle cellular components in eukaryotic cells to maintain the stability of the intracellular environment and the cells ability to cope with unfavorable environments. Numerous studies suggest that autophagy participates in regulating various cellular functions and is closely associated with the onset and progression of various diseases. Wound healing is a complex, multistep biological process that involves multiple cell types. Refractory wounds, which include diabetic skin ulcers, can seriously endanger human health. Previous studies have confirmed that autophagy plays an essential role in various phases of wound healing. Specifically, in the inflammatory phase, autophagy has an anti-infection effect and it negatively regulates the inflammatory response, which prevents excessive inflammation from causing tissue damage. In the proliferative phase, local hypoxia in the wound can induce autophagy, which plays a role in anti-apoptosis and anti-oxidative stress and promotes cell survival. Autophagy of vascular endothelial cells promotes wound angiogenesis and that of keratinocytes promotes their differentiation, proliferation and migration, which is conducive to the completion of wound re-epithelialisation. In the remodeling phase, autophagy of fibroblasts affects the formation of hypertrophic scars. Additionally, a refractory diabetic wound may be associated with increased levels of autophagy, and the regulation of mesenchymal stem cell autophagy may improve its application to wound healing. Therefore, understanding the relationship between autophagy and skin wound healing and exploring the molecular mechanism of autophagy regulation may provide novel strategies for the clinical treatment of wound healing.

Icariin activates autophagy to trigger TGFβ1 upregulation and promote angiogenesis in EA.hy926 human vascular endothelial cells

ABSTRACT Angiogenesis plays an important role in tissue development and repair, and how to regulate angiogenesis effectively is a widely studied problem in the biomedical field. In recent years, the role of autophagy in vascular endothelial cells has attracted extensive attention. Icariin (ICA) is a traditional Chinese medicine that has been proven to have outstanding protective effects on the vascular system and to regulate cellular autophagy effectively. However, at present, it has not been reported whether ICA can affect the angiogenic ability of endothelial cells by affecting autophagy. In this study, we aimed to investigate whether ICA affects the angiogenesis capacity of EA.hy926 human vascular endothelial cells through autophagy and explain the underlying potential mechanisms. First, we determined that ICA at appropriate concentrations has the ability to promote cell migration and angiogenesis using wound healing assays and tube formation assays. Then, at the molecular level, we observed the upregulation of VEGFA, VEGFR2, ANGI, ANGII, and Tie2 mRNA and detected the upregulation of TGFβ1 protein by Western blotting. We also demonstrated that angiogenic concentrations of ICA can effectively activate autophagy. The autophagy inhibitor 3-MA significantly suppressed TGFβ1 expression and tube formation in EA.hy926 cells. Overall, we hope that our studies might help to further understand the effect of ICA on vascular endothelial cells and provide a theoretical basis for future angiogenic applications of ICA

Resolvin D1 Suppresses H2O2-Induced Senescence in Fibroblasts by Inducing Autophagy through the miR-1299/ARG2/ARL1 Axis.

ARG2 has been reported to inhibit autophagy in vascular endothelial cells and keratinocytes. However, studies of its mechanism of action, its role in skin fibroblasts, and the possibility of promoting autophagy and inhibiting cellular senescence through ARG2 inhibition are lacking. We induced cellular senescence in dermal fibroblasts by using H2O2. H2O2-induced fibroblast senescence was inhibited upon ARG2 knockdown and promoted upon ARG2 overexpression. The microRNA miR-1299 suppressed ARG2 expression, thereby inhibiting fibroblast senescence, and miR-1299 inhibitors promoted dermal fibroblast senescence by upregulating ARG2. Using yeast two-hybrid assay, we found that ARG2 binds to ARL1. ARL1 knockdown inhibited autophagy and ARL1 overexpression promoted it. Resolvin D1 (RvD1) suppressed ARG2 expression and cellular senescence. These data indicate that ARG2 stimulates dermal fibroblast cell senescence by inhibiting autophagy after interacting with ARL1. In addition, RvD1 appears to promote autophagy and inhibit dermal fibroblast senescence by inhibiting ARG2 expression. Taken together, the miR-1299/ARG2/ARL1 axis emerges as a novel mechanism of the ARG2-induced inhibition of autophagy. Furthermore, these results indicate that miR-1299 and pro-resolving lipids, including RvD1, are likely involved in inhibiting cellular senescence by inducing autophagy.

Identification of a new autophagy inhibitor targeting lipid droplets in vascular endothelial cells

Autophagy of vascular endothelial cells (VECs) plays an important role in maintaining vascular homeostasis. Lipid droplets (LDs) are organelles that can be formed in response to various stimuli, including excessive lipid or various stresses. LDs sequester toxic lipids, thereby preventing lipotoxic cell damage and have a complex relationship with autophagy. In the previous study, we identified a novel Grp94 inhibitor HCP1 inhibited apoptosis in VECs. Here we found that HCP1 targeted LDs and promoted the accumulation of LDs in VECs. Our results showed that HCP1 upregulated the protein levels of autophagy-related proteins. We demonstrated that HCP1 upregulated the number of LDs and suppressed autophagy by inhibiting Grp94. Therefore, we provided HCP1 as a new VECs autophagy inhibitor targeting LDs, which might be a potential compound in the treatment of VECs autophagy related vascular diseases.

Different effects of anti-VEGF drugs (Ranibizumab, Aflibercept, Conbercept) on autophagy and its effect on neovascularization in RF/6A cells

IntroductionChoroidal neovascularization (CNV) is the main pathological change of wet age-related macular degeneration. Anti-VEGF drugs are the most commonly used treatment for CNV. The biggest drawback of anti-VEGF drugs is the recurrence of CNV, which requires repeated therapy several times. Autophagy activation may be involved in reducing the therapeutic effect of anti-VEGF drugs. So, this study aims to elucidate the effect and mechanism of anti-VEGF drugs on endothelial autophagy and neovascularization in vitro. MethodsRF/6A cells were randomly divided into five groups: The control group, hypoxia group (1% O2, 5% CO2, 94% N2), anti-VEGF group (group1: Ranibizumab 100 μg/ml; group2: Aflibercept, 400 μg/ml; group3: Conbercept, 100 μg/ml). Autophagy-related proteins were examined by Western blot. RFP-GFP-LC3 was used to detect autophagy and autophagic flow. Subsequently, we used autophagy inhibitors (3-MA or CQ) to inhibit Conbercept induced autophagy and to observe its effect on angiogenesis in vitro. Proliferation, migration, and tube formation of endothelial cells can be used to study neovascularization in vitro. In this research, the CCK-8 assay was used to detect cell proliferation. Cell migration and tube formation were assessed by wound assay and matrix method, respectively. Flow cytometry and Tunel were used to detect cell apoptosis. Finally, the mechanism of Conbercept activated autophagy was studied. Western blot was used to detect the expression of p53 and DRAM (damage-regulated autophagy modulator), upstream activators of autophagy. ResultsThe protein levels of Beclin-1 and LC3-2/1 in Ranibizumab and Conbercept groups were significantly higher than in the hypoxia group(P < 0.05). While the expression of P62 was decreased (P < 0.05). The autophagic flux was showed the same results. However, Aflibercept showed the opposite effect on autophagy. Compared with the Conbercept group, autophagy inhibitor 3-MA or CQ can further inhibit cell proliferation and promotes cell apoptosis (P < 0.05). Conbercept significantly inhibited cell migration compared with the hypoxia group (633.08 ± 72.52 vs. 546.33 ± 24.61), while the autophagy inhibitor group (3-MA or CQ) had a more obvious inhibition effect (309.75 ± 86.36 and 263.33 ± 68.67) (P < 0.05). For tube formation, the number of tube formation was decreased significantly in the Conbercept group (32.00 ± 2.00) compared to the hypoxia group (39.00 ± 1.53) and even further reduced in 3-MA or CQ group (24.00 ± 3.61, 20.00 ± 2.65). The length of master segments in the hypoxia group was 15,668.00 ± 894.11. It was decreased in Conbercept (13,885.34 ± 730.03). In 3-MA or CQ group, the length of master segments dropped further (11,997.00 ± 433.66, 10,617.67 ± 543.21). Compare with the hypoxia group, the expression P53 and DRAM were increased in the Conbercept group (P < 0.05). Autophagy-related proteins LC-3, Beclin-1, and DRAM were inhibited by P53 inhibitor Pifithrin-α (PFTα) (P < 0.05). ConclusionRanibizumab and Conbercept can trigger the autophagy of vascular endothelial cells while Aflibercept can inhibit it. The combination of Conbercept and autophagy inhibitor can significantly inhibit the formation of angiogenesis in vitro. The mechanism of autophagy activation is related to the activation of the p53/DRAM pathway.

Excess fibronectin 1 participates in pathogenesis of pre-eclampsia by promoting apoptosis and autophagy in vascular endothelial cells.

Plasma fibronectin 1 (FN1) levels are elevated in individuals with pre-eclampsia (PE), which may be applied as a possible b marker for vascular endothelial injury during PE. In the present study, the possible role of FN1 in the pathogenesis of PE and regulation of apoptosis and autophagy in vascular endothelial cells was explored. Plasma FN1 levels in 80 patients with PE and 40 healthy pregnant individuals were measured using ELISA to verify its relationship with the severity of PE. pcDNA3.1-FN1 or FN1-small interfering (si) RNA was used to manipulate the expression of FN1 in human umbilical vein endothelial cells (HUVECs) to assess the effects of FN1 on cell apoptosis, autophagy, and the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) signaling pathway. It was found that upregulation of FN1 promoted apoptosis and autophagy, in addition to significantly inhibiting the activation of AKT and mTOR in HUVECs. By contrast, downregulation of FN1 expression inhibited cell apoptosis and autophagy, but increased AKT and mTOR phosphorylation in HUVECs that were cultured in serum samples obtained from patients with PE. Rescue experiments found that the PI3K/AKT inhibitor LY294002 reversed the effects of FN1-siRNA on apoptosis and autophagy in HUVECs cultured in serum from patients with PE. Therefore, data from the present study suggest that FN1 participates in the pathogenesis of PE by promoting apoptosis and autophagy in vascular endothelial cells, which is associated with the PI3K/AKT/mTOR signaling pathway.

Autophagy Induced by a Novel Triazol Derivative Promotes Angiogenesis Through Decreasing Interferon-Inducible Protein 10 Level in Vascular Endothelial Cells.

Autophagy plays an important role in angiogenesis, whereas the mechanisms of vascular endothelial cell (VEC) autophagy associated with angiogenesis remain unclear. In this study, we identified a novel triazol derivative (JL025) that significantly promoted angiogenesis both in vitro and in vivo. Moreover, JL025 had no effects on cell proliferation but dramatically increased the autophagy level of VEC. The suppression of autophagy inhibited JL025-induced angiogenesis in vitro and in vivo, suggesting that JL025-induced angiogenesis was dependent on the enhanced autophagy. Mechanistic studies indicated that JL025-induced VEC autophagy was related to the Protein Kinase B/mTOR signaling pathway. Meanwhile, JL025 decreased the antiangiogenic chemokine interferon-inducible protein 10 (IP10) protein level in human VECs. Importantly, the suppression of autophagy inhibited JL025-induced decrease of IP10 protein level, indicating that autophagy mediated the degradation of IP10. Taken together, our findings provide new insights into the relationship of VEC autophagy with angiogenesis, and JL025 may have a therapeutic potential in related diseases.

Loss of GRB2 associated binding protein 1 in arteriosclerosis obliterans promotes host autophagy.

Background:Arteriosclerosis obliterans (ASO) is a major cause of adult limb loss worldwide. Autophagy of vascular endothelial cell (VEC) contributes to the ASO progression. However, the molecular mechanism that controls VEC autophagy remains unclear. In this study, we aimed to explore the role of the GRB2 associated binding protein 1 (GAB1) in regulating VEC autophagy.Methods:In vivo and in vitro studies were applied to determine the loss of adapt protein GAB1 in association with ASO progression. Histological GAB1 expression was measured in sclerotic vascular intima and normal vascular intima. Gain- and loss-of-function of GAB1 were applied in VEC to determine the effect and potential downstream signaling of GAB1.Results:The autophagy repressor p62 was significantly downregulated in ASO intima as compared to that in healthy donor (0.80 vs. 0.20, t = 6.43, P < 0.05). The expression level of GAB1 mRNA (1.00 vs. 0.24, t = 7.41, P < 0.05) and protein (0.72 vs. 0.21, t = 5.97, P < 0.05) was significantly decreased in ASO group as compared with the control group. Loss of GAB1 led to a remarkable decrease in LC3II (1.19 vs. 0.68, t = 5.99, P < 0.05), whereas overexpression of GAB1 significantly led to a decrease in LC3II level (0.41 vs. 0.93, t = 7.12, P < 0.05). Phosphorylation levels of JNK and p38 were significantly associated with gain- and loss-of-function of GAB1 protein.Conclusion:Loss of GAB1 promotes VEC autophagy which is associated with ASO. GAB1 and its downstream signaling might be potential therapeutic targets for ASO treatment.

Substrate stiffness differentially impacts autophagy of endothelial cells and smooth muscle cells

Stiffening of blood vessels is one of the most important characteristics in the process of many cardiovascular pathologies such as atherosclerosis, angiosteosis, and vascular aging. Increased stiffness of the vascular extracellular matrix drives artery pathology and alters phenotypes of vascular cell. Understanding how substrate stiffness impacts vascular cell behaviors is of great importance to the biomaterial design in tissue engineering, regenerative medicine, and medical devices. Here we report that changing substrate stiffness has a significant impact on the autophagy of vascular endothelial cells (VECs) and smooth muscle cells (VSMCs). Interestingly, our findings demonstrate that, with the increase of substrate stiffness, the autophagy level of VECs and VSMCs showed differential changes: endothelial autophagy levels reduced, leading to the reductions in a range of gene expression associated with endothelial function; while, autophagy levels of VSMCs increased, showing a transition from contractile to the synthetic phenotype. We further demonstrate that, by inhibiting cell autophagy, the expressions of endothelial functional gene were further reduced and the expression of VSMC calponin increased, suggesting an important role of autophagy in response of the cells to the challenge of microenvironment stiffness changing. Although the underlying mechanism requires further study, this work highlights the relationship of substrate stiffness, autophagy, and vascular cell behaviors, and enlightening the design principles of surface stiffness of biomaterials in cardiovascular practical applications.

Naringin inhibits autophagy mediated by PI3K-Akt-mTOR pathway to ameliorate endothelial cell dysfunction induced by high glucose/high fat stress

As a flavonoid, naringin (Nar) has been shown to have multiple pharmacological effects including lowering blood cholesterol, reducing thrombus formation and improving microcirculation. However, effects of Nar on function and autophagy of vascular endothelial cells under high glucose and high fat (HG/HF) stress are largely unclear. This study was designed to investigate such effects of Nar in human umbilical vein endothelial cells (HUVECs) and to determine whether such effects are related to autophagy. Our present results show that 86 μM of Nar inhibits the autophagy levels and protects the cells against the dysfunction induced by HG/HF stress. Moreover, Nar increases the phosphorylation levels of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt) and mammalian rapamycin target protein (mTOR). However, pretreatment with rapamycin (RAPA, 5 μM, autophagy inducer), LY294002(10 μM, PI3K inhibitor) and Akt inhibitor Ⅳ (0.5 μM, Akt inhibitor) partially abrogates the protective effects of Nar, suggesting that the protective effects of Nar are achieved by activating the PI3K-Akt-mTOR pathway to inhibit autophagy. In conclusion, Nar improves the function of HUVECs under HG/HF stress through activating the PI3K-Akt-mTOR pathway to inhibit autophagy. The findings offer an insight into HG/HF stress-induced autophagy and indicate that Nar might have potential to prevent and treat the diabetic angiopathy.