RESUMO
OBJECTIVE: To study the effects of inhibited Annexin A2 (ANXA2) on human umbilical vein endothelial cells (HUVECs) in vitro. METHODS: Short hairpin RNA (shRNA) targeting ANXA2 was designed and cloned into double marked lentivirial vector GV248 for RNAi to generate the recombinant expression plasmids, which were stably transfected into HUVECs. The protein and mRNA expression levels of ANXA2 were analyzed by western blotting and real-time polymerase chain reaction, respectively. Cell proliferation (cell counting kit-8 assay), apoptosis (flow cytometry analysis), the expression (western blotting) and the activity of caspases (enzyme-linked immunosorbent assay) were used to assess the effects of silencing ANXA2 on HUVECs in vitro. RESULTS: The plasmids to express ANXA2-specific shRNA were constructed and were infected into HUVEC resulting in the stably transfected experimental (ANXA2-shRNA), control (control-shRNA) and mock (no plasmid) cell lines, which were verified with western blot and real-time PCR. HUVEC/ANXA2-shRNA showed an inhibition rate 91.89% of ANXA2 expression compared to the mock HUVEC. ANXA2 silencing cell strain obviously presented a lower cell proliferation activity compared to the control and mock HUVECs, with an inhibition rate 82.35% on day 7 in vitro. FACS analysis indicated that the HUVEC/ANXA2-shRNA cells undergoing apoptosis increased by 102.61% compared to the mock HUVECs (P < 0.01). Moreover, the activity levels of caspase-3, caspase-8 and caspase-9 in HUVEC/ANXA2-shRNA cells were increased and the activated cleaved caspase-3, cleaved caspase-8 and cleaved caspase-9 were upregulated evidently compared with that of the control and mock HUVECs by 56.29%, 89.59% and 144.58% (P < 0.01). CONCLUSIONS: shRNA-mediated silencing of ANXA2 could not only be able to suppress HUVECs proliferation but to upregulate the enzyme activity of caspases, which bring to an increase of cell apoptosis. This work suggested that ANXA2 may represent a useful target of future molecular therapies.
RESUMO
BACKGROUND: Choroidal neovascularization (CNV) is a common cause of visual loss in the elderly patients with age-related macular degeneration and represents the growth of subretinal new vessels in the macular region. This study aimed to investigate the relationship between annexin A2 (ANXA2) and vascular endothelial growth factor (VEGF) in CNV. METHODS: In a rat model of argon laser coagulation-induced CNV, the mRNA expressions of the annexins and VEGF protein expression in the retina were detected using fluorescent real-time polymerase chain reaction (PCR) and immunohistochemistry, respectively. The interactions between ANXA2 and VEGF in both a retinal pigment epithelial cell line RPE-J and the rat model of CNV were examined by means of RNA interference, real-time PCR, Western blotting, enzyme-linked immunosorbent assay (ELISA) and histopathological examinations. RESULTS: Fundus fluorescein angiography (FFA) showed that argon laser coagulation of the retina induced stable CNV models in the rats. Two to three weeks after the coagulation, ANXA2 and VEGF expressions in the coagulated area in the retina and choroid increased to the peak level, while the other annexin members (ANXA4, ANXA5, ANXA7 and ANXA11) showed no obvious changes. In RPE-J cells and the CNV model, RNA interference of ANXA2 gene significantly lowered the VEGF protein and mRNA expressions, and application of an adenoviral vector containing ANXA2 gene markedly increased VEGF expressions in the rat model of CNV, but produced no significant effects on the expressions of the kinase insert domain-containing receptor (KDR) or the fms-like tyrosine kinase (Flt-1). The expression of KDR inhibited the increment in ANXA2 expression, but VEGF and Flt-1 did not directly affect ANXA2 expression. CONCLUSION: Besides the role as a plasminogen and the receptor of tissue plasminogen activator, ANXA2, which is under regulation of KDR via a negative feedback mechanism, also participates in neovascularization by regulating VEGF expression through a positive feedback mechanism.
