The Study Of Vacuolation And Association Between Apoptosis And Senescence In Vacular Endothelial Cells By A Novel Butyrolactone Derivate

Vascular endothelial cells (VECs) form the inner lining of all blood vessels and function to maintain vascular tone and anticoagulant properties of vessels. VEC dysfunction could be an important mechanism of various cardiovascular diseases. Thus we tried to study the relationship between apoptosis and senescence and the mechanism of vacuolation in human umbilical vascular endothelial cells (HUVECs).Our previous study has found that the deprivation of serum and FGF-2 induced apoptosis in HUVECs. But whether it could induce senescence simultaneously is unknown. In this study, we found that the deprivation of serum and FGF-2 induced HUVECs senescence as well as apoptosis. In the light of chemical genetics, we detected the key factors involved in the association of apoptosis and senescence using a new derivative of butyrolactone (3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-one,3BDO)Vacuolation is a common event in apoptosis, necrosis. It might be correlated with cell senescence. Thus it is of great importance to study the mechanism of vacuolation. Our previous study demonstrated that Chloroquine (CQ) at lower concentrations could induce vacuolation in A549 lung cancer cells. In this study, we dected the effects of CQ in HUVECs and found that CQ (8μM to 32μM) induced vacuolation with increased volume of acidic compartments (VAC) in VECs in a dose-dependent manner. In order to find novel agents that affect vacuolation induced by CQ, we sequentially investigated the effects of 3BDO on the vacuolation in HUVECs.METHODS:1. HUVECs were gained as described previously [Jaffe et al., 1973]. 2. The morphological changes were observed under phase contrast microscope.3. The cell proliferation was measured by MTT assay.4. DNA nuclear fragmentation was analyzed by acridine orange staining.5. The TdT-mediated dUTP nick end labeling technique was used to detect in situ nuclear DNA fragmentation and count apoptosis ratio.6. The senescent status of cells was analyzed by in situ staining for SA-8-galactosidase.7. The expression and distribution of integrin β4 was analysised by immunofluorescence assay.8. The fluorescence probe, DCHF was used to analyze ROS level.9. The lysossomal vacuolation degree-the volume of acidic compartments (VAC) was measured by neutral red uptake method.10. Na~+K~+-ATPase activity assay was performed by the Na~+K~+-ATPase detection kit.RESULTS:1. Deprivation of FGF-2 not only induced apoptosis, but also triggered senescence in HUVECs.2. 3BDO (60-180μM) inhibited the apoptosis and senescence by deprivation of serum and FGF-2 simultaneously.3. The high level of integrin β4 induced by the deprivation of serum and FGF-2 could be inhibited by the treatment of 3BDO.4. 3BDO could not affect the accumulation of intracellular ROS.5. CQ (8μM-32μM) induced vacuolation in HUVECs in a dose-dependent manner.6. 3BDO (60μM-180μM) inhibited the vacuolation induced by CQ with the decreased volume of acidic compartments (VAC) in HUVECs.7. Na~+K~+-ATPase activity was decreased during the vacuolation induced by CQ. 3BDO elevated the low level of Na~+K~+-ATPase activity induced by CQ.CONCLUSION:1. In this study, we found that besides apoptosis, senescence was also triggered by deprivation of serum and FGF-2 in HUVECs. The result is in line with the notion that cellular senescence and apoptosis coincide in parallel.2. We first discovered a new small molecule, 3BD0, that had anti-apoptotic and anti-senescent effects on vascular endothelial cells. The finding offered us an exciting tool to study the relationship between apoptosis and senescence.3. Integrin 64 was implicated in the anti-apoptotic and anti-senescent effects of 3BD0, suggesting that integrin 64 might be a key factor in the signal cross-talking between cellular senescence and apoptosis.4. 3BD0 could not depress the ROS level although it inhibited the apoptosis and senescence at the same time. These data suggested that there were ROS-independent pathways that mediated apoptosis and senescence in HUVECs.5. CQ as a vacuolation inducer and 3BDO as a vacuolation inhibitor in HUVECs offered us a pair of exciting tools to study the mechanism of cellular vacuolation.6. Na~+K~+-ATPase might be involved in the process that 3BD0 inhibited the vacuolation induced by CQ.