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Published in final edited form as: Circulation. 2012 Dec 25;127(3):365–376. doi: 10.1161/CIRCULATIONAHA.112.118174

Uremic serum stabilizes tissue factor (TF) by inhibiting its ubiquitination, and indole-3-acetic acid (IA) and indoxyl sulfate (IS) recapitulate its effect on TF. A, Both nondiabetic and diabetic uremic sera increase TF half-life. Vascular smooth muscle cells (vSMCs) treated with pooled 5% control or nondiabetic or diabetic uremic sera for 24 hours are shown. Emetine 20 μmol/L was used for indicated time. The cells were harvested, and TF expression was analyzed by immunoblotting. Representative blot from 3 experiments is shown. Densitometry was performed on half-life study with the use of Image J, and pancadherin was used to normalize the TF signal. Average of 3 experiments is shown. *P value is 0.03 for both the DM and non-DM sera treated vSMCs compared to the control. Error bars=SEM. DM indicates diabetes mellitus. B, Uric acid does not affect half-life of TF. vSMCs treated with NaOH and uric acid for 24 hours were treated with emetine as above. Representative blot from 3 experiments is shown. Densitometry was performed on half-life study with Image J, and pancadherin was used to normalize the TF signal. *P value is 0.02 for both the IA and IS treated vSMCs compared to the control. Average of 3 experiments is shown. Error bars=SEM. C, IA and IS prolong half-life of TF. vSMCs exposed to protein-bound solutes and control (treated with human serum albumin [HSA]) for 24 hours were treated with emetine for indicated time. The cells were harvested, and TF expression was analyzed by immunoblotting. Representative blot from 3 experiments is shown. Densitometry was performed on half-life study shown in Figure 4F with the use of Image J, and pancadherin was used to normalize the TF signal. *P value is 0.02 for both the IA and IS treated vSMCs compared to the control. Average of 3 experiments is shown. Error bars=SEM. D, Ubiquitination of endogenous TF. vSMCs 1·10⁶ were treated with MG132. TF was immunoprecipitated (IP) with the use of TF antibody and immunoblotted with ubiquitin antibodies. Immunoprecipitated TF is shown as input. Representative immunoblot from 3 experiments is shown. Densitometry analysis was performed on ubiquitin smear normalized with immunoprecipitated TF with the use of Image J. Average of 3 experiments is shown. P<0.05 for ubiquitin without vs with MG132. Error bars=SEM. E, Uremic serum inhibits TF ubiquitination. vSMCs treated with 5% pooled control or nondiabetic or diabetic uremic sera for 24 hours were lysed after treatment with MG132. TF ubiquitination was examined as described above. Representative immunoblot from 3 experiments is shown. Densitometry analysis was performed on ubiquitin smear as described above. Average of 3 experiments is shown. P<0.05 for non-DM and DM uremic compared with control serum. Error bars=SEM. F, Water-soluble uremic solutes have no effect on TF ubiquitination. vSMCs were treated with uremic solutes for 24 hours and 10 μM MG132 for the last 6 hours. Water-treated sample served as control. Representative immunoblot from 2 experiments is shown. Densitometry analysis was performed as described above. Average of 2 experiments is shown. Error bars=SEM. G, IA and IS inhibit TF ubiquitination. vSMCs were treated with protein-bound uremic solutes or uric acid solutes for 24 hours and MG132. TF was immunoprecipitated as above. Human albumin or NaOH served as control. Representative immunoblot from 3 experiments is shown. Densitometry analysis was performed as described above. Average of 3 experiments is shown. P<0.05 for IA and IS compared with human serum albumin (control). Error bars=SEM. DMSO indicates dimethyl sulfoxide; and WB, westernblot.

Uremic serum stabilizes tissue factor (TF) by inhibiting its ubiquitination, and indole-3-acetic acid (IA) and indoxyl sulfate (IS) recapitulate its effect on TF. A, Both nondiabetic and diabetic uremic sera increase TF half-life. Vascular smooth muscle cells (vSMCs) treated with pooled 5% control or nondiabetic or diabetic uremic sera for 24 hours are shown. Emetine 20 μmol/L was used for indicated time. The cells were harvested, and TF expression was analyzed by immunoblotting. Representative blot from 3 experiments is shown. Densitometry was performed on half-life study with the use of Image J, and pancadherin was used to normalize the TF signal. Average of 3 experiments is shown. *P value is 0.03 for both the DM and non-DM sera treated vSMCs compared to the control. Error bars=SEM. DM indicates diabetes mellitus. B, Uric acid does not affect half-life of TF. vSMCs treated with NaOH and uric acid for 24 hours were treated with emetine as above. Representative blot from 3 experiments is shown. Densitometry was performed on half-life study with Image J, and pancadherin was used to normalize the TF signal. *P value is 0.02 for both the IA and IS treated vSMCs compared to the control. Average of 3 experiments is shown. Error bars=SEM. C, IA and IS prolong half-life of TF. vSMCs exposed to protein-bound solutes and control (treated with human serum albumin [HSA]) for 24 hours were treated with emetine for indicated time. The cells were harvested, and TF expression was analyzed by immunoblotting. Representative blot from 3 experiments is shown. Densitometry was performed on half-life study shown in Figure 4F with the use of Image J, and pancadherin was used to normalize the TF signal. *P value is 0.02 for both the IA and IS treated vSMCs compared to the control. Average of 3 experiments is shown. Error bars=SEM. D, Ubiquitination of endogenous TF. vSMCs 1·10⁶ were treated with MG132. TF was immunoprecipitated (IP) with the use of TF antibody and immunoblotted with ubiquitin antibodies. Immunoprecipitated TF is shown as input. Representative immunoblot from 3 experiments is shown. Densitometry analysis was performed on ubiquitin smear normalized with immunoprecipitated TF with the use of Image J. Average of 3 experiments is shown. P<0.05 for ubiquitin without vs with MG132. Error bars=SEM. E, Uremic serum inhibits TF ubiquitination. vSMCs treated with 5% pooled control or nondiabetic or diabetic uremic sera for 24 hours were lysed after treatment with MG132. TF ubiquitination was examined as described above. Representative immunoblot from 3 experiments is shown. Densitometry analysis was performed on ubiquitin smear as described above. Average of 3 experiments is shown. P<0.05 for non-DM and DM uremic compared with control serum. Error bars=SEM. F, Water-soluble uremic solutes have no effect on TF ubiquitination. vSMCs were treated with uremic solutes for 24 hours and 10 μM MG132 for the last 6 hours. Water-treated sample served as control. Representative immunoblot from 2 experiments is shown. Densitometry analysis was performed as described above. Average of 2 experiments is shown. Error bars=SEM. G, IA and IS inhibit TF ubiquitination. vSMCs were treated with protein-bound uremic solutes or uric acid solutes for 24 hours and MG132. TF was immunoprecipitated as above. Human albumin or NaOH served as control. Representative immunoblot from 3 experiments is shown. Densitometry analysis was performed as described above. Average of 3 experiments is shown. P<0.05 for IA and IS compared with human serum albumin (control). Error bars=SEM. DMSO indicates dimethyl sulfoxide; and WB, westernblot.