Effects And Mechanisms Of Angiopoietins On Mouse Glomerular Endothelial Cell Senescence

PART I The established model of mouse glomerular endothelial cell senescence induced by hydrogen peroxideOBJECTIVE:Normal primary cells proliferate a limited number of times in vitro and activate senescence process due to progressive telomere shortening. Furthermore, cells may also undergo acute stress-induced senescence, which could be triggered by cumulative stimuli involving oxidation, ultraviolet(UV) light, toxic compounds, as well as oncogens. Stress induces senescence through telomere-independent mechanisms, and displays all the major characteristics of replicatively senescent cells. The molecular basis of aging in organs is not well established, but organ aging may reflect aspects of cellular senescence involved with molecular pathway changes. In recent years, much attention has been devoted to studying the relevance of cellular senescence to aging and disease. H2O2, a common causative agent of cellular senescence, can produce various types of DNA damage, which represents the type of stress-induced senescence via DNA damage and cannot be rescued by over-expression of telomerase reverse transcriptase(TERT).The purpose of the study is to investigate whether H2O2 can induce mouse glomerular endothelial cells(MGECs) senescence in vitro.METHODS:The MGECs stimulated with hydrogen peroxide(H2O2) were cultured in vitro,and were devided into control group,H2O2 group. The senescent status of cells were verified by senescence-associated P-Galactosidase (SA-β-Gal) staining. The cell cycle of mouse glomerular endothelial cells was analyzed by flow cytometry. The expression of p16 was detected by Western Blot. The senescent of MGECs which was induced by H2O2, was evaluated according above detection. Furthermore, the apoptosis characteristic of MGECs was observed by Hoechst-33342 staining and flow cytometry. RESULTS:1. MGECs treated with H2O2 (50μM) for 1 week exhibited an enlarged size and flattened morphology, characteristic of the senescent phenotype of MGECs.2. The staining of SA-β-Gal assay show that:there was almost negative expression of SA-P-Gal activity in control group; The effect that H2O2 increased SA-β-gal-positive cells was increased with longer incubating time.With H2O2 treatment, the percentage of SA-β-gal-positive MGECs was increased from 13.12%at 3 days to 70.47%at 7 days.3. Through analyzing from cell cycle result, we could conclude that:compared with control group, H2O2-induced senescent cells presented with the classical senescence-related cell cycle dysfunction. H2O2-induced senescence group mitosis is delayed in G1 phase and the cells have a limited capacity to enter S phase (P＜0.05).4. Western blot data demonstrated that H2O2 induced the significant increasing in induction of p16INK4a(Control:21.09±0.67 vs. H2O2:69.39±4.10).5. After hoechest-33342 staining, we could see no apoptosis morphology changes. Furthermore,the result of flow cytometry didn't show typical sub-G1 apoptosis peak. H2O2 did not induce apoptosis (apoptosis rate:3.19±0.49% vs 3.26±0.39%, P>0.05).CONCLUSIONS:H2O2 can induce mouse glomerular endothelial cells senescence in vitro.MGECs treated with H2O2 (50μM) for 1 week exhibited characteristic of the senescent phenotype of MGECs, increased SA-β-gal-positive cells and the expression of p16INK4a presented with cell cycle G1 arrest. Furthermore, H2O2 did not induce apoptosis. These results indicate that H2O2 can mimic the induction of the cell senescent phenotype. PART II Effects of Angiopoietins on the senescence of mouse glomerular endothelial cell induced by hydrogen peroxideOBJECTIVE:Aging is a normal biological process defined as a form of permanent and irreversible proliferation arrest, with progressive organ modification and ultimately the loss of organ function. The kidney also participates in the aging process of the whole body, and age-associated changes have many implications for nephrology, including the slightly lower level of renal function with normal aging. The vasculature plays a key role in the progression of renal damage in aging, with reduction in glomerular and peritubular capillary density and decreased endothelial proliferative response. However, a direct link between changes in renal capillary endothelium and aging-associated renal disease has not been yet elucidated. The angiopoietins, which regulate the transition between a mature stable vasculature and angiogenic or remodeling vessels, play an important role in vascularization. Angl acts primarily as an agonist ligand of Tie2, which induces Tie2 phosphorylation, stabilizes vascular branch networks, and promotes endothelial cell survival. In contrast, Ang2 is a natural antagonist ligand of Tie2, promoting vascular destabilization by opposing the effects of Ang1. In the second part of this study, we investigated the role of Angl in protecting mouse glomerular endothelial cells from H2O2-induced cellular senescence.,and discuss its preliminary mechanisms.METHODS:The MGECs were cultured in vitro and devided into control group,H2O2 group,Ang1 group Ang2 group. MGECs were subjected to H2O2 induced senescence, which was evaluated by senescence-associated (3-Galactosidase (SA-P-Gal) staining, cell-cycle analysis and expression of p16. Endothelial cell function was assessed by NO, vWF secretion and capillary-like structure formation. The assays of malonaldehyde (MDA),glutathione(GSH),superoxide dismutase (SOD) and catalase (CAT) in cell lysis were measured by colorimetric method. Western blot were used to locate and quantitate the signal activity of Tie2,ERK1/2.RESULTS:1. Senescent phenotypes:The percentage of SA-β-gal-positive MGECs was increased with H2O2 treatment. Co-incubation with Angl markedly inhibited the induction of senescence by 51.80%(P<0.05). However, SA-β-Gal activity of MGECs treated with H2O2 in the presence of Ang2 showed no significant change. H2O2 group mitosis is delayed and the cells have a limited capacity to enter S phase. In contrast, Angl treatment induced a dramatic change in this pattern, the G1 population reduced from 84.10%incubated with H2O2 to 74.50%, whereas S populations were increased from 10.88% to 18.89%. No significant difference in this pattern of distribution was observed in cells treated with only Ang2. H2O2 induced the increased induction of p16INK4a, Angl showed 44.30% down-regulated p16INK4a levels in MGECs under H2O2 stimulation. However, under Ang2 treatment conditions, high expression of p16INK4a was also noticeable.2. Endothelial cell functions:The mean NO level decreased dramatically in senescence cells in contrast to control. Whereas incubation of MGECs with Angl resulted in a significant increase in the release of NO(15.61±1.61 vs 10.26±0.69, P<0.05). However, there was no significant difference in NO concentration between Ang2 and H2O2 group. Elevated levels of vWF were detected in the culture medium of senescent cells.Treatment of endothelial cells with Angl had a decreased effect on release of vWF into the culture medium(5.13±0.69 vs 9.88±0.53, P<0.05). In contrast, when endothelial cells were treated with Ang2, no difference was detected in the amount of vWF secreted by the senescent cells (Ang2:10.02±0.31, P>0.05) Angl-treated cells formed capillary-like structures more efficiently than senescent cells at base line. In contrast, no significant changes were observed with Ang2.3. Oxidative stress:MDA of H2O2 group was significantly higher than that of control group. Whereas incubation of MGECs with Angl resulted in a significant decrease in the release of MDA(H2O2; 10.31±0.22 vs. Ang1; 5.02±0.47, P＜0.05). In contrast, when endothelial cells were treated with Ang2, no difference was detected in the amount of MDA. SOD,GSH and CAT of H2O2 group were notably lower in contrast to control. Treatment of endothelial cells with Angl had a increase effect on activity of SOD,GSH and CAT(P<0.05). No significant difference was observed in cells treated with only Ang2(P>0.05).4. Tie2-ERKl/2 signaling:Western blot data demonstrated that a dramatic increase of Tie2 tyrosine phosphorylation was detected exposure to Angl, while total Tie2 level was not affected(H2O2:4.12±2.06 vs. Ang1: 71.09±2.15, P<0.05). In contrast, in the H2O2 plus Ang2 group, there was no significant elevation of Tie2 phosphorylation level (Ang2:16.07±3.16, P>0.05). Angl caused significant elevation of phosphorylated but not total-ERK levels(P<0.05). However, Ang2 could not elicit ERK activation.CONCLUSIONS:The present studies suggest that Angl inhibits H2O2-induced senescence in mouse glomerular endothelial cells。Moreover, Angl regulated the secretion and capillary-like structure formation of endothelial cells with aging. Improvement of oxidative stress may also be involved. Angl causes significant elevation of phosphorylated Tie2 and ERK1/2 levels. However, under Ang2 treatment conditions, no significant difference was observed in cells treated with H2O2. PARTⅢThe mechanisms of angiopoietin-1 inhibits mouse glomerular endothelial cell senescenceMETHODS:The MGECs were cultured in vitro and devided into control group,H2O2 group,Ang1 group,Ang2+Ang1 group,sTie2-Fc group,PD98059 group. MGECs were subjected to H2O2 induced senescence, which was evaluated by senescence-associatedβ-Galactosidase (SA-β-Gal) staining, cell-cycle analysis and expression of p16. Endothelial cell function was assessed by NO, vWF secretion and capillary-like structure formation. Western blot were used to locate and quantitate the signal activity of Tie2,ERK1/2.OBJECTIVE:Angl acts primarily as an agonist ligand of Tie2, which induces ERK1/2 phosphorylation, stabilizes vascular branch networks, and promotes endothelial cell survival. In the second part of this study, we have found Ang1could significant elevated phosphorylation of Tie2 and ERK1/2 levels. However the question arose as to whether Tie2-ERK1/2 signal was involved in the inhibition of cellular senescence induced by Ang1 in MGECs. To further investigated the signaling pathway of Tie2 in mediating the effect of ERK1/2 in mouse glomerular endothelial cells during aging, cells were pretreated with Ang2, Tie2 antagonist (sTie2-Fc) or a specific ERK1/2 inhibitor(PD98059) before adding Ang1.RESULTS:1. Senescent phenotypes:Compaired with Ang1, the percentage of SA-β-gal-positive MGECs was increased with Ang2,sTie2-Fc and PD98059 treatment(Ang2:63.94±2.05, sTie2-Fc:72.56±1.51, PD98059:73.31±2.73 vs. Ang1:18.67±2.73, P＜0.05). MGECs treated with sTie2-Fc, Ang2 and PD98059 subsequently exhibited the same senescence-associated morphologies as H2O2-induced senescence cells. Cell cyle is delayed and the cells have a limited capacity to enter S phase. Angl showed down-regulated p16INK4a levels in MGECs under H2O2 stimulation. However, such effects could be completely eliminated either by sTie2-Fc, Ang2 or by PD98059(Ang2:58.39±2.02, sTie2-Fc:61.08±3.10, PD98059:62.19±3.72vs. Ang1:21.16±1.11, P<0.05).2. Endothelial cell functions:Angl resulted in a significant increase in the release of NO. However, this change was completely blocked by treatment with Ang2, sTie2-Fc and PD98059(Ang2:11.92±0.44, sTie2-Fc:10.22±0.36, PD98059:10.87±0.66 vs. Ang1:16.38±0.35, P＜0.05). Treatment of endothelial cells with Ang2, sTie2-Fc and PD98059 had a increased effect on release of vWF into the culture medium (P<0.05). Cells incubated with Ang2, sTie2-Fc or PD98059 plus Angl failed to stimulate capillary-like structure formation(Ang2:23.11±2.29, sTie2-Fc:15.21±3.31, PD98059:18.56±2.36 vs.Ang1:57.66±3.17, P<0.05).3. Tie2-ERK1/2 signaling:Western blot data demonstrated that sequestering Angl by Ang2 and sTie2-Fc blocked ERK phosphorylation(Ang2: 47.12±2.89, sTie2-Fc:35.62±3.71 vs. Angl:135.16±2.27, P<0.05). Furthermore, when a specific ERK inhibitor PD98059 was used in combination with Ang1, the same intense bands corresponding to phosphorylation of Tie2 tyrosine were observed as cells treated with Ang1(Ang1:61.25±1.91, vs.PD98059:63.72±2.87, P>0.05).CONCLUSIONS:The present studies suggest that Angl inhibits H2O2-induced senescence in mouse glomerular endothelial cells via the Angl-Tie2-ERKl/2 signaling pathway.