Hyperphosphatemia induces protective autophagy in endothelial cells through the inhibition of Akt/mTOR signaling  Yu-Juei Hsu, MD, PhD, Shih-Che Hsu, MSc, Shih-Ming Huang, PhD, Herng-Sheng Lee, MD, PhD, Shih-Hua Lin, MD, Chien-Sung Tsai, MD, Chun-Che Shih, MD, PhD, Chih-Yuan Lin, MD  Journal of Vascular Surgery  Volume 62, Issue 1, Pages 210-221.e2 (July 2015) DOI: 10.1016/j.jvs.2014.02.040 Copyright © 2015 Society for Vascular Surgery Terms and Conditions

Fig 1 Hyperphosphatemia is associated with increased endothelial autophagy in chronic kidney disease (CKD) rats. A, Serum phosphate (Pi) and creatinine levels in 5/6 nephrectomy (CKD) and sham-operated rats. Representative immunofluorescence images (1000× original magnification) double-stained with microtubule-associated protein 1 light chain 3 (LC3, red) and von Willebrand factor (green) (B) and immunofluorescence staining of cleaved caspase 3 (green) (C) in the aortic endothelial cells of sham-operated and CKD rats. Nuclei were visualized by DAPI staining (blue). The data are presented as the means ± standard error of the mean (n = 8). Semiquantification of fluorescence intensity of LC3 (D) and cleaved caspase 3 (E) staining in the aortic endothelial cells of sham-operated and CKD rats. *P < .05 vs sham-operated rats. Red, blue, and white arrows indicate LC3-positive, LC3-negative, and cleaved caspase 3–positive endothelial cells, respectively. Journal of Vascular Surgery 2015 62, 210-221.e2DOI: (10.1016/j.jvs.2014.02.040) Copyright © 2015 Society for Vascular Surgery Terms and Conditions

Fig 2 Dose- and time-course for high phosphate (Pi)-induced autophagy in endothelial cells in vitro. Representative immunoblots of protein samples collected from cell lysates of Pi-treated human microvascular endothelial cell (HMEC-1) cells labeled with antibodies against microtubule-associated protein 1 light chain 3 (LC3) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH). A, Dose course of LC3 expression in HMEC-1 cells treated with the indicated concentration of Pi for 24 hours, and cells cultured in medium containing 1 mM Pi were used as the control. B, Time course of LC3 expression in HMEC-1 cells treated with Pi (3 mM) for the indicated durations, and cells collected at time zero were used as the control. C, Autophagy activity, as measured through the LC3-II/LC3-I ratio, was quantified through computer-assisted densitometry analysis and presented through relative percentages compared with the corresponding controls. D-G, Representative confocal microscopy images of immunofluorescence staining for LC3 (green) in HMEC-1 cells treated with the indicated concentration of Pi for 24 hours (D) or transfected with green fluorescent protein (GFP)–LC3 plasmids, cultured for 6 hours, and then treated with Pi (3 mM) for the indicated durations (F). The autophagosomes are detected as green fluorescence puncta. Semiquantification of fluorescence intensity of LC3 staining in HMEC-1 cells treated with the indicated concentration of Pi for 24 hours (E) or 3 mM Pi for the indicated durations after transfection with GFP-LC3 plasmids (G). The nuclei were visualized by DAPI staining (blue). The data are presented as the means ± standard error of the mean (n = 3). *P < .05 vs the corresponding control. #P < .05 vs cells treated with 2 mM Pi for 24 hours or 3 mM Pi for 12 hours. Journal of Vascular Surgery 2015 62, 210-221.e2DOI: (10.1016/j.jvs.2014.02.040) Copyright © 2015 Society for Vascular Surgery Terms and Conditions

Fig 3 High phosphate (Pi) induces the inhibition of Akt/mammalian target of rapamycin (mTOR) signaling in a dose- and time-dependent manner. Representative immunoblots of protein samples collected from cell lysates of Pi-treated human microvascular endothelial cell (HMEC-1) cells labeled with antibodies against Akt phosphorylated at Ser 473 (p-Akt), Akt, mTOR phosphorylated at Ser 2448 (p-mTOR), mTOR, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). A, Dose course of Akt and mTOR phosphorylation in HMEC-1 cells treated with the indicated concentration of Pi for 24 hours, and cells cultured in medium containing 1 mM Pi were used as the control. B, Time course of Akt and mTOR phosphorylation in HMEC-1 cells treated with Pi (3 mM) for the indicated durations, and cells collected at time zero were used as the control. C, The levels of Akt and mTOR phosphorylation were quantified through computer-assisted densitometry analysis, and levels of phosphoprotein immunoreactivity were normalized to their respective total protein immunoreactivity and expressed as percentages of the control. The data are presented as the means ± standard error of the mean (n = 3). *P < .05 vs the corresponding control. Journal of Vascular Surgery 2015 62, 210-221.e2DOI: (10.1016/j.jvs.2014.02.040) Copyright © 2015 Society for Vascular Surgery Terms and Conditions

Fig 4 Blockage of phosphate (Pi) influx reverses high Pi-induced autophagy and the inhibition of Akt/mammalian target of rapamycin (mTOR) signaling in endothelial cells. A, Human microvascular endothelial cell (HMEC-1) cells were pretreated with PiT-1 inhibitor (phosphonoformic acid [PFA]) for 1 hour and then with Pi (3 mM) for 24 hours. The cell lysates were analyzed by immunoblotting with antibodies against microtubule-associated protein 1 light chain 3 (LC3), Akt phosphorylated at Ser 473 (p-Akt), Akt, mTOR phosphorylated at Ser 2448 (p-mTOR), and mTOR. The LC3-II/LC3-I ratio (B) and the levels of Akt (C) and mTOR phosphorylation (D) were quantified by computer-assisted densitometry analysis, and the levels of phosphoprotein immunoreactivity were normalized to their respective total protein immunoreactivity and expressed as percentages of the control. E, Representative images for the immunofluorescence staining of LC3 (green) in HMEC-1 cells treated with Pi (3 mM) for 24 hours with or without PFA (1 mM) before treatment. The nuclei were visualized by DAPI staining (blue). The data are presented as the means ± standard error of the mean (n = 3). *P < .05 vs the corresponding control. #P < .05 vs cells treated with 3 mM Pi. GAPDH, Glyceraldehyde 3-phosphate dehydrogenase. Journal of Vascular Surgery 2015 62, 210-221.e2DOI: (10.1016/j.jvs.2014.02.040) Copyright © 2015 Society for Vascular Surgery Terms and Conditions

Fig 5 Blockage of autophagy exacerbates high phosphate (Pi)-induced apoptosis in endothelial cells. A, Representative immunoblots of microtubule-associated protein 1 light chain 3 (LC3), cleaved caspase 3, and cleaved poly(ADP-ribose) polymerase (PARP) in human microvascular endothelial cell (HMEC-1) cells exposed to Pi (3 mM) with 3-methyladenine (3-MA; 5 mM) or rapamycin (Rapa; 0.1 mM) for 24 hours. B, The expression levels of cleaved caspase 3 and PARP protein were quantified by computer-assisted densitometry analysis, are normalized to the β-actin expression levels, and are expressed as relative percentages compared with the corresponding control. The data are presented as the means ± standard error of the mean (n = 3). *P < .05 vs the corresponding control. #P < .05 vs cells treated with 3 mM Pi. †P < .05 vs cells treated with 3 mM Pi + 3-MA. Journal of Vascular Surgery 2015 62, 210-221.e2DOI: (10.1016/j.jvs.2014.02.040) Copyright © 2015 Society for Vascular Surgery Terms and Conditions

Fig 6 Flow cytometry analysis of the effect of high phosphate (Pi)-induced autophagy on endothelial cell apoptosis. A-E, Flow cytometry analysis of apoptosis by annexin V–phycoerythrin (PE) and 7-amino-actinomycin D (7-ADD) staining. Human microvascular endothelial cell (HMEC-1) cells were exposed to Pi (3 mM) and cotreated with 3-methyladenine (3-MA; 5 mM) or rapamycin (Rapa; 0.1 mM). After 24 hours of incubation, the cells were harvested and analyzed with an imaging cytometer. The dots with annexin V−/7-ADD+ (Q1, left upper quadrant), annexin V+/7-AAD+ (Q2, right upper quadrant), annexin V−/7-ADD− (Q3, left lower quadrant), and annexin V+/7-ADD− (Q4, right lower quadrant) features represent dead, late-apoptotic or necrotic, viable, and early apoptotic cells, respectively. F, The percentage of early apoptotic cells (Q4) was calculated and is shown in the histogram as the relative fold change compared with the corresponding control. The data are presented as the means ± standard error of the mean (n = 3). *P < .05 vs the corresponding control. #P < .05 vs cells treated with 3 mM Pi. †P < .05 vs cells treated with 3 mM Pi + 3-MA. Journal of Vascular Surgery 2015 62, 210-221.e2DOI: (10.1016/j.jvs.2014.02.040) Copyright © 2015 Society for Vascular Surgery Terms and Conditions

Supplementary Fig 1 (online only) High phosphate (Pi) induces the inhibition of Akt/mammalian target of rapamycin (mTOR) signaling in a time-dependent manner. Representative immunoblots of protein samples collected from cell lysates of Pi-treated Human microvascular endothelial cell (HMEC-1) cells labeled with antibodies against Akt phosphorylated at Ser 473 (p-Akt), Akt, mTOR phosphorylated at Ser 2448 (p-mTOR), mTOR, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). A, Time course of Akt and mTOR phosphorylation in HMEC-1 cells treated with Pi (3 mM) for the indicated durations (0.25 h, 0.5 h, 1 h, 3 h, 6 h), and cells collected at time zero were used as the control. B, The levels of Akt and mTOR phosphorylation were quantified through computer-assisted densitometry analysis, and levels of phospho-protein immunoreactivity were normalized to their respective total protein immunoreactivity and expressed as percentages of the control. The data are presented as the means ± standard error of the mean (n = 3). *P < .05 versus the corresponding control. Journal of Vascular Surgery 2015 62, 210-221.e2DOI: (10.1016/j.jvs.2014.02.040) Copyright © 2015 Society for Vascular Surgery Terms and Conditions

Supplementary Fig 2 (online only) Blockage of phosphate (Pi) influx reverses high-Pi-induced autophagy and the inhibition of Akt/mammalian target of rapamycin (mTOR) signaling in endothelial cells. A, Human microvascular endothelial cell (HMEC-1) cells were pretreated with PiT1 inhibitor (phosphonoformic acid [PFA]) or LY294002 for 1 h and then with Pi (3 mM) for 24 h. The cell lysates were analyzed by immunoblotting with antibodies against microtubule-associated protein 1 light chain 3 (LC3), Akt phosphorylated at Ser 473 (p-Akt), Akt, mTOR phosphorylated at Ser 2448 (p-mTOR), and mTOR. The LC3-II/LC3-I ratio (B) and the levels of Akt (C) and mTOR phosphorylation (D) were quantified by computer-assisted densitometry analysis, and the levels of phospho-protein immunoreactivity were normalized to their respective total protein immunoreactivity and expressed as percentages of the control. The data are presented as the means ± standard error of the mean (n = 3). *P < .05 versus the corresponding control; #P < .05 versus cells treated with 3 mM Pi; †P < .05 versus cells treated with 3 mM Pi + PFA. LY294002, Akt inhibitor. Journal of Vascular Surgery 2015 62, 210-221.e2DOI: (10.1016/j.jvs.2014.02.040) Copyright © 2015 Society for Vascular Surgery Terms and Conditions