Annexin A2 silencing enhances apoptosis of human umbilical vein endothelial cells in vitro

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.

Annexin A2 silencing enhances apoptosis of human umbilical vein endothelial cells in vitro

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.

Annexin A2 promotes choroidal neovascularization by increasing vascular endothelial growth factor expression in a rat model of argon laser coagulation-induced choroidal neovascularization / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Quantification of posterior capsular opacification after cataract surgery / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To evaluate posterior capsular opacification (PCO) with hydrophobic acrylic intraocular lens (IOL, Sensar AR40e) and silicone IOLs after cataract surgery, to use a software program developed to provide an objective assessment of the amount of PCO in the digital images of the posterior capsule to quantify PCO. METHODS: Ninety-eight eyes underwent standardized phacoemulsification and "in the bag" IOL placement, were randomized to receive a three piece lens of hydrophobic acrylic or silicone, but lens materials were different in one case. In year 1 and 2, digitized retro-illumination images were taken from the posterior capsule. Images were analyzed by POCO software program, removing the Purkinje light reflexes, contrast enhancement, filtering to enhance low-density PCO. RESULTS: The percentage of PCO were 0.32±0.13 of hydrophobic acrylic IOLs in year 1, compared with 0.39±0.17 of silicone (P=0.37). In year 2, the percentage of PCO were 0.42±0.20 with hydrophobic acrylic IOLs and 0.34±0.18 with silicone IOLs (P=0.50). Of those patients with PCO in year 1 and 2, severity grades were 0.50±0.30 and 0.82±0.58 of hydrophobic acrylic cases, compared with 0.63±0.35 and 0.55±0.35 of patients with silicone IOLs (P=0.52,P=0.69) with no statistical significance.CONCLUSION: The POCO system is capable of producing an objective and repeatable measure of PCO that is relevant to assessing techniques of PCO prevention.

Management of primary angle-closure glaucoma by phacoemulsification with foldable posterior chamber intraocular lens implantation / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To evaluate the efficacy and safety of phacoemulsification with foldable posterior chamber intraocular lens(PC-IOL) implantation in the management of acute or chronic primary angle-closure glaucoma(APACG or CPACG) with cataract.</p><p><b>METHODS</b>Twenty-six cases(28 eyes) with simple cataract and 60 cases(70 eyes)of PACG with cataract, including 14 eyes of preclinical APACG, 29 eyes of paroxysmal APACG, 27 eyes of CPACG. Visual acuity distributed from hand movement to 0.8. Phacoemulsification and intraocular lens implantation were performed and patients were followed up for(8.5+/-4.5) months.</p><p><b>RESULT</b>In simple cataract group, postoperative IOP did not decrease(t=1.9201, P>0.05); in preclinical APACG group, postoperative IOP decreased (t=3.9910, P<0.01); in paroxysmal APACG group, postoperative IOP decreased (t=4.7441, P<0.01); in CPACG groups, postoperative IOP decreased (t=4.4976, P<0.01). In APACG and CPACG groups postoperative antiglaucoma medication reduced. In APACG and CPACG groups, angle of anterior chamber was widen. Preoperative central and peripheral ACD of APACG and CPACG were much less than those of postoperative. In 56 eyes of PACG, visual acuity was corrected in 48 eyes(85.7%) including 28 eyes(50%) with corrected visual acuity>0.5.</p><p><b>CONCLUSION</b>The phacoemulsification with foldable posterior chamber intraocular lens implantation is beneficial for PACG with cataract, and the curative efficacy of APACG is better than that of CPACG.</p>

Combined lens and vitreoretinal surgery in patients with traumatic cataract and intraocular foreign body / 国际眼科杂志(Guoji Yanke Zazhi)

·AIM: To analyze the postoperative anatomical and functional outcomes as well as complications after combined phacoemulsification, pars plana vitrectomy (PPV), removal of the intraocular foreign body (IOFB) and intraocular lens (IOL) implantation in patients with traumatic cataract and intraocular foreign body.·METHODS: Medical records of 13 patients (13 eyes)with traumatic cataract and IOFB who had undergone combined phacoemulsification, PPV, foreign body extraction and IOL implantation were retrospectively analyzed. The postoperative follow-up ranged from 2 to 12 months. The main measurements of outcomes were the extraction success of cataract and intraocular foreign body, intraoperative and postoperative complications and the final best corrected visual acuity (BCVA).·RESULTS: The mean age of 13 patients (10 male, 3 female ) was 36.8 years (range: 17-65 years). All eight lOFBs were removed. Four intraocular lenses were implanted after vitrectomy intraoperatively. In 5 cases, intraocular lenses were implanted during the second operation. Intraocular lenses were not implanted in 4 cases. BCVA at last ranged from 0.8 to hand movement. BCVA was 0. 5 or better in four eyes, 0.1 to 0. 4 in five eyes, less than 0.1 in four eyes. Intraoperative complications were encountered in 3 patients. They had vitreous hemorrhage. Postoperative complications were encountered in 2 patients. They had retinal detachment. The reoperations of the two patients were successful.·CONCLUSION; The combined phacoemulsification,PPV, removal of IOFB and IOL implantation is safe and effective for patients with traumatic cataract and intraocular foreign body. The visual outcome depended primarily on the corneal or scleral wound and underlying posterior segment pathology and sites.