2023 Volume 46 Issue 11 Pages 1512-1516
Vascular endothelial cells sustain vascular health through barrier and endocrine functions. Insufficient oxygen supply induces endothelial dysfunction in the pathology of various diseases. In addition, oxygen deprivation reportedly induces endothelial dysfunction via autophagy. Ras guanyl-releasing protein 2 (RasGRP2) has guanosine 5′-diphosphate (GDP)/guanosine 5′-triphosphate (GTP) exchange factor activity and activates Rap1 and R-Ras which belong to the small GTPases. RasGRP2 exerts protective effects against vascular endothelial dysfunction. However, the effect of RasGRP2 on hypoxic stress in vascular endothelial cells has not yet been investigated. We examined the protein expression of hypoxia-inducible factor (HIF)-1α, BCL2 interacting protein 3 (BNIP3), and microtubule-associated protein light chain 3β (LC3β). We observed that oxygen deprivation increased the expression of HIF-1α, BNIP3 and LC3β II. RasGRP2 suppressed the induction of HIF-1α and the subsequent increase in LC3β II. These findings suggest the possibility that RasGRP2 plays a protective role against endothelial dysfunction by suppressing oxygen deprivation-induced autophagy.
Vascular endothelial cells form a monolayer in the innermost layer of blood vessels and are involved in the secretion of biologically active substances, such as nitric oxide (NO) and hormones, as well as in the formation of a barrier between the blood and tissues, contributing to the health of blood vessels. Studies have reported that damage to vascular endothelial cells due to the loss of these functions is associated with various diseases, including lifestyle-related diseases such as arteriosclerosis, hypertension, and diabetes.1,2) Notably, insufficient oxygen supply is associated with the pathology of diseases such as arteriosclerosis, ischemia, diabetic retinopathy, and sleep apnea syndrome.3–5)
Autophagy, which is a homeostatic mechanism that decomposes intracellular proteins and organelles, is induced through hypoxia-inducible factor (HIF)-1α and BCL2 interacting protein 3 (BNIP3) in hypoxic environments.6) In general, autophagy has protective effects on cells.7) Reentry, the negative effects of autophagy was revealed gradually. Qian et al. reported that autophagy caused by decreased expression of Grb2-associated binder 1 impairs endothelial function in human umbilical vein endothelial cells (HUVECs).8) In addition, Zhang et al. reported that RAGE-mediated autophagy promotes endothelial-mesenchymal transition in vascular endothelial cells.9) Metformin, an antidiabetic agent, improves hyperglycemia-induced endothelial damage through down-regulation of autophagy.10) Furthermore, autophagy induction by oxygen and glucose deprivation treatment reportedly damages the vascular barrier in the blood–brain barrier.11) Consequently, autophagy is a key factor in endothelial dysfunction. However, comprehensive therapeutic approaches for controlling the autophagy in endothelial dysfunction have not been developed, and more detailed studies on the regulation of autophagy in endothelial cells are required.
Ras guanyl releasing protein 2 (RasGRP2) is a guanine nucleotide exchange factor and its function has been studied in human platelets, leukocytes, and vascular endothelial cells.12) RasGRP2 expressed in vascular endothelial cells is involved in Xenopus angiogenesis. We previously reported the increase of spatiotemporal expression of RasGRP2 was observed in Xenopus vascular development.13–15) Furthermore, we reported that RasGRP2 activates Ras-associated protein 1 (Rap1) and Ras related protein (R-Ras), which belong to the group of small guanosine 5′-triphosphatases (GTPases), and inhibits apoptosis of human vascular endothelial cells through the activation of Akt, which plays a critical role in cell survival signaling via R-Ras, and the suppression of reactive oxygen species (ROS) generation via Rap1.16,17) RasGRP2 inhibits vascular hyperpermeability by toxic advanced glycation end products (TAGE) through these signaling pathways.18) As a result, RasGRP2 may play key roles in maintaining vascular health through cytoprotective effects mediated by Rap1 and R-Ras. However, the function of RasGRP2 in hypoxic environments remains unclear. In this study, the effect of RasGRP2 on autophagy by oxygen deprivation treatment was investigated using telomerase reverse transcriptase human umbilical vein endothelial cells (TERT HUVEC) stably overexpressing RasGRP2.
Chloroquine (CQ) was purchased from Cayman Chemical (14194, U.S.A.). CQ dissolved in dimethyl sulfoxide (DMSO) (25 µM final concentration) was used to inhibit autophagy.
Cell CultureTERT HUVECs were kindly provided by Dr. Kazuto Nishio (Kindai University).18) TERT HUVECs were cultured in an endothelial cell growth medium (C-22010, PromoCell, Germany) at 5% CO2 and 37 °C. A stable RasGRP2-overexpressing cell line was previously established.18) The amplified DNA fragment of rasgrp2 was transfected into TERT HUVEC using pEB Multi-Hyg (050-08121, FUJIFILM Wako Pure Chemical Corporation, Japan) as a vector and ViaFect Transfection Reagent (E4981, Promega, U.S.A.) as a transfection reagent.
Cells were seeded in culture dishes (2.0 × 105 cells/mL) and incubated for 6 d (culture medium was changed every 2 d) before each experiment.
Oxygen Deprivation TreatmentThe ischemic pathology was mimicked by oxygen deprivation treatment. Oxygen deprivation treatment (0% O2) was conducted using the BIONIX-1 hypoxic culture kit (SUGIYAMA-GEN CO., LTD., Japan) after 6 d of incubation.
Preparation of Cell Lysate and Western Blotting (WB)After normoxic or oxygen deprivation treatment, the cells were lysed in Pierce™ IP lysis buffer (87787, Thermo Fisher Scientific Inc., U.S.A.) containing Halt™ protease and phosphatase inhibitor single-use cocktail (78442, Thermo Fisher Scientific Inc.). The cell lysate was centrifuged at 12000 × g for 10 min. After centrifugation, the supernatants were examined by WB. The cell lysate was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to an Immobilon®-P polyvinylidene difluoride (PVDF) membrane (IPVH00010, Millipore, Germany). Membranes were incubated with PVDF Blocking Reagent for Can Get Signal® (NYPBR01, Toyobo, Japan) for 1 h, and then with the following primary antibodies diluted in Can Get Signal Solution 1 (NKB-201, Toyobo) for 1 h at room temperature: rabbit anti-RASGRP2 (1 : 10000; GTX108616, GeneTex, U.S.A.), mouse anti-HIF-1α (1 : 1000; sc-53546, Santa Cruz Biotechnology, Inc., U.S.A.), mouse anti-microtubule-associated protein light chain 3β (LC3β, 1 : 1000; sc-376404, Santa Cruz Biotechnology, Inc.), mouse anti-BNIP3 (1 : 1000; sc-56167, Santa Cruz Biotechnology, Inc.), and rabbit anti-α-tubulin (1 : 10000; GTX112141, GeneTex). This was followed by incubation with horseradish peroxidase-conjugated secondary antibodies, anti-rabbit immunoglobulin G (IgG) (1 : 200000; NB7179, Novus Biologicals, U.S.A.), and anti-mouse IgG (1 : 5000; P0260, Dako, Denmark), diluted in Can Get Signal Solution 2 (NKB-301, Toyobo) for 1 h at room temperature, Immunoreactive bands were detected. The immunoreactive band was quantified using ImageJ.
Statistical AnalysisAll experiments were performed at least three times. The data are presented as the mean ± standard deviation. Differences between two groups were analyzed using one-way ANOVA and unpaired Student’s t-tests. The level of statistical significance was set at p < 0.05.
The protein level of RasGRP2 was analyzed to confirm the overexpression of RasGRP2 protein in the RasGRP2 stable overexpression (R) cells. Compared to Mock (M) cells, the RasGRP2 protein level was highly increased in R cells. On the long exposure time, the band of the RasGRP2 was observed in M cells (Fig. 1).
Western blotting analysis of RasGRP2 in M and R cells. The images of RasGRP2 were obtained at short (1 s) and long (10 min) exposure times. M, mock cells; R, RasGRP2 stable overexpression cells.
To investigate the effect of RasGRP2 on oxygen deprivation-induced autophagy, the protein expression levels of HIF-1α, LC3β, and BNIP3 were analyzed by WB. In M cells, the expression level of HIF-1α, a marker of hypoxia, increased significantly under oxygen deprivation treatment at 3–12 h and decreased thereafter. In R cells, the expression level of HIF-1α increased significantly under oxygen deprivation treatment for 3 and 9 h. However, the HIF-1α expression levels in R cells were significantly lower than those in M cells at 6 and 12 h (Fig. 2B). The oxygen deprivation treatment increased the ratio of LC3β II/I, an indicator of autophagy, significantly, at 6–24 h in M cells. In R cells, the ratio of LC3β II/I was increased significantly in the 6, 9, and 24 h oxygen deprivation treatments. The increase in the ratio of LC3β II/I was significantly attenuated in R cells when compared to that in M cells (Fig. 2C) after 6 and 9 h oxygen deprivation treatments. The protein expression level of BNIP3, which is involved in the induction of autophagy in oxygen deprivation, was increased significantly after 6–12 h of oxygen deprivation treatment in M cells. Oxygen deprivation treatment for 6–9 h increased the expression of BNIP3 in R cells. The induction of BNIP3 by oxygen deprivation treatment was significantly attenuated in R cells when compared to that in M cells after 6–9 h oxygen deprivation treatment (Fig. 2D).
(A) Western blot analysis of HIF-1α, microtubule-associated protein light chain 3β (LC3β), BCL2 interacting protein 3 (BNIP3) and α-tubulin protein expression after each oxygen deprivation treatment (0% O2) time. The quantification of immunoreactive bands; (B) HIF-1α, (C) LC3β, and (D) BNIP3. Zero hour indicates the time before oxygen deprivation treatment. The data are presented as mean ± standard deviation (S.D.). n = 3; statistical significance is denoted by * p < 0.05, compared with M at each time, # p < 0.05, compared with 0 h. M, mock cells; R, RasGRP2 stable overexpression cells.
To examine autophagosome formation in both cells, both cells were pretreated with CQ, an autophagy inhibitor, and the accumulation of LC3β II during oxygen deprivation treatment (24 h) was analyzed by WB. The pretreatment with CQ (25 µM, 30 min) accumulated LC3β II in both cells (Fig. 3A). Compared with the normoxic treatment (21% O2, 24 h), oxygen deprivation treatment significantly increased the ratio of LC3β II/I in both cells. Pretreatment with CQ significantly increased the ratio of LC3β II/I compared to pretreatment without CQ in both cells (Fig. 3B).
(A) Western blot analysis of LC3β and α-tubulin protein expression after each treatment (21 or 0% O2, for 24 h). Cells were pretreated with or without CQ (25 µM) 30 min prior to oxygen deprivation treatment (0% O2). The data are presented as mean ± S.D. n = 3; statistical significance is denoted by # p < 0.05, compared with each 21% O2, + p < 0.05, compared with each 0% O2 without CQ. M, mock cells; R, Ras guanyl releasing protein 2 stable overexpression cells.
The HIF-1α expression level is regulated by synthesis and degradation. The synthesis of HIF-1α is activated by phosphatidylinositol-3 kinase/Akt signaling.19) In contrast, HIF-1α is degraded by proteasomes through proline hydroxylation by the prolyl hydroxylase domain-containing protein (PHD2) and ubiquitination by ubiquitin ligase complexes, including the von Hippel–Lindau protein (pVHL) in normoxia. These enzymes are inactivated, and the HIF-1α expression level increases in hypoxic environments. pVHL is activated by Akt,20) and PHD2 is inactivated by ROS.21) HIF-1α levels in vascular endothelial cells increase according to the duration of exposure to hypoxia and then decrease after reaching a peak.22,23) Furthermore, we previously reported that RasGRP2 inhibits ROS production and promotes Akt phosphorylation.16,17) In this study, RasGRP2 decreased the protein expression level of HIF-1α without changing the HIF-1α expression pattern depending on the duration of exposure to the oxygen deprivation (Fig. 2B). This finding indicated that RasGRP2 decreases the HIF-1α expression levels by favoring the degradation system of HIF-1α as a result of the combined effects of the suppression of the inactivation of PHD2 by ROS inhibition and the enhancement of the degradation system through the activation of pVHL by Akt and synthesis system through the activation of Akt. To elucidate the detailed mechanism of the enhancement of the degradation system of HIF-1α by RasGRP2, it is necessary to analyze the proline hydroxylation and ubiquitination of HIF-1α.
Autophagy in a hypoxic environment is induced by the release of Beclin-1 through increased expression of HIF-1α and BNIP3.6) However, phosphorylation of Akt inhibits autophagy.24) With the progression of autophagy, phosphatidylethanolamine is added to LC3β II. The expression level of LC3β II is widely used to evaluate autophagy because it is involved in the formation of an isolation membrane and subsequent formation of autophagosome.25) Notably, oxygen deprivation treatment increased the expression levels of LC3β II and BNIP3, which are indicators of autophagy, in both cell types, but the increase in LC3β II and BNIP3 was lower in R cells compared to M cells (Figs. 2C, D). This suggests that RasGRP2 inhibits autophagy in hypoxic environments by phosphorylating Akt and suppressing HIF-1α/BNIP3 expression. BNIP3 induced in a hypoxic environment replaces Beclin-1 complexed with Bcl-XL and Bcl-2, thereby releasing Beclin-1.26) Released Beclin-1 is involved in the induction of autophagy; therefore, it is necessary to analyze the release of Beclin-1 from the complex with Bcl-XL and Bcl-2 in the future.
CQ, a representative autophagy inhibitor, inhibit the fusion of autophagosome and lysosome.27) CQ suppresses the degradation of LC3β II in autolysosomes and accumulates LC3β II in autophagosomal membranes.28) CQ pretreatment is used to evaluate the autophagosome formation. LC3β II accumulation by CQ reflects autophagy occurred for the treatment duration. In the present study, similar LC3β II accumulation was observed in both cell types after oxygen deprivation with CQ pretreatment (Fig. 3). Therefore, RasGRP2 inhibits the oxygen deprivation-induced autophagy without affecting basal autophagosome formation ability.
HIF-1α is related to cell survival under hypoxia.29) Knockdown of HIF-1α induced the apoptosis in endothelial cells.30) Therefore, accurate assessment of the oxygen deprivation-induced autophagy is difficult due to the influence of cell death. Further research is required to reveal the mechanisms in detail.
Recently, the negative effect of autophagy on endothelial cells has gained significant attention and is under investigation.31–33) However, the quantitative relationship between autophagy and endothelial dysfunction is difficult to evaluate. Thus, additional investigation is required to elucidate whether RasGRP2 prevents vascular endothelial dysfunction.
Our study demonstrated RasGRP2 attenuated oxygen deprivation-induced increase in HIF-1α, BNIP3, and LC3β II. RasGRP2 may exert a protective effect against vascular endothelial dysfunction caused by autophagy in hypoxia environment, such as arteriosclerosis and ischemia.
This research was supported by JSPS KAKENHI Grant Number: JP22K17789.
The authors declare no conflict of interest.
