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Targeting STUB1–tissue factor axis normalizes hyperthrombotic uremic phenotype without increasing bleeding risk

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Science Translational Medicine  22 Nov 2017:
Vol. 9, Issue 417, eaam8475
DOI: 10.1126/scitranslmed.aam8475
  • Fig. 1. IS mediates a hyperthrombotic uremic phenotype in an AHR-dependent manner across CKD stages.

    (A) Mean blood concentrations of IS in 10- to 14-week-old male and female C57BL/6 mice (n = 5 per time point) are shown. The P values correspond to an increase in IS concentrations compared to the probenecid group. No differences in blood concentrations of IS were noted between male and female mice. The dashed line represents the average concentration of IS in ESRD patients (11, 47). Student’s t test was performed. Data are shown as means ± SD. (B) Representative traces of carotid artery flow in C57BL/6 animals before and 20 min after the application of 10% FeCl3 (n = 8 per group). The time for the blood flow to drop below 0.299 ml/min was considered as TtO (arrow). (C) Representative images from hematoxylin and eosin–stained carotid arteries collected after the FeCl3 procedure. Representative images from six vessels per group are shown. Scale bars, 25 μm. RT, right; LT, left. (D) Mean TtO (n = 8 per group) after 5 days of exposure to IS and IS + CH223191. Student’s t test was performed. Data are shown as means ± SD. (E) Mice were exposed to probenecid + IS for 5 days and then subjected to the FeCl3 thrombosis assay. Thirty minutes before the assay, control antibody or rat anti-mouse TF neutralizing antibody was administered. Mean TtO (n = 6 per group) is shown. Data are shown as means ± SEM. (F) Mean TtO in the photochemical thrombosis model in probenecid and probenecid + IS mice (n = 6 per group). Student’s t test was performed. Data are shown as means ± SEM. (G) Relationship between TtO and different concentrations of IS in 16 animals. IS concentrations from different stages of human CKD are shown (3, 11). A Pearson correlation analysis was performed.

  • Fig. 2. STUB1 destabilizes and ubiquitinates TF and mediates TF regulation by AHR.

    (A) Lysates from primary human aortic vSMCs transfected with control (Csi) or STUB1 silencing oligonucleotides (STUB1si) were probed for TF and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using ImageJ. Equal amounts of lysates were separately probed for STUB1 to avoid stripping of blots for the proteins with molecular weights in close range, a strategy also applied to other figures. Expression of TF and STUB1 normalized to the loading control is shown below this and subsequent Western blots. A representative figure from four independent experiments is shown. (B) vSMCs pretransfected with Csi and STUB1si were stimulated with 5% of the indicated type of serum for 24 hours. A mean TF activity of two independent experiments performed in duplicates is shown. Student’s t test was performed. Number sign (#) corresponds to TF activity in Csi vSMCs treated with uremic compared to control serum. Other P values correspond to STUB1-silenced compared to Csi cells. Data are shown as means ± SD. (C) vSMCs pretransfected with Csi and STUB1si grown in 5% uremic serum for 24 hours and then treated with cycloheximide (80 μM) to inhibit protein translation for the indicated time. Equal amounts of lysates were probed separately to confirm STUB1 silencing. TF expression normalized to the loading control is depicted below the blot. A representative figure from four independent experiments is shown. (D) Densitometry of normalized TF expression is represented as the percentage of TF at time 0. The time to reach 50% of initial TF was considered as the half-life of TF. An average of four experiments is shown. Data are shown as means ± SD. (E) vSMCs pretransfected with Csi and STUB1si were treated with 5% uremic serum with or without the AHR antagonist CB7993113 (20 μM) or vehicle for 24 hours. A representative of two independent experiments performed in duplicate is shown. (F) vSMCs pretransfected with Csi and STUB1si were treated with 5% uremic serum + 20 μM CB7993113 (12 hours) and 10 μM MG132 (4 hours) before harvest. The lysates immunoprecipitated with anti-TF antibody were probed with anti–ubiquitin (Ub) antibody. Five percent of lysates are shown as inputs. A representative of three independent experiments is shown. IP, immunoprecipitation; WB, Western blotting.

  • Fig. 3. A dynamic STUB1-TF interaction depends on the uremic status.

    (A) Recombinant GST-tagged STUB1 protein was immobilized on glutathione S-transferase (GST) beads and treated with lipidated human recombinant TF (rhTF) protein for 4 hours. Eluents were probed for bound TF. Five percent of recombinant GST-tagged STUB1 stained with Coomassie and recombinant TF are shown as inputs. Representative immunoblots from four independent experiments are shown. (B) vSMCs were pretreated with 10 μM IS (24 hours) and 20 μM CH223191 (20 min) followed by immunoprecipitation using anti-STUB1 antibody, and the eluents were probed for TF. The stripped blot was reprobed for STUB1. Five percent of cell lysates are shown as inputs. Representative blots from three independent experiments are shown. DMSO, dimethyl sulfoxide. (C) Human embryonic kidney (HEK) 293T cells stably expressing FLAG-tagged WT TF (TF-WT) or TF C-terminal truncation (TFdelC) were transfected with myelocytomatosis (MYC)–tagged STUB1. Cell lysates were immunoprecipitated with FLAG or MYC antibodies, and coimmunoprecipitated proteins were detected using MYC and FLAG antibodies. Five percent of lysates were probed as inputs. A representative of two independent experiments done in duplicates is shown. (D) HEK293T cells coexpressing FLAG-tagged WT TF and empty vector (control) or MYC-tagged STUB1 plasmids were treated with cycloheximide for the indicated amount of time. The TF bands were normalized to GAPDH. Equal amounts of lysates were probed for MYC-tagged STUB1. A representative of three independent experiments is shown. (E) HEK293T cells coexpressing FLAG-tagged TFdelC and empty vector (control) or MYC-tagged STUB1 were processed as in (D). A representative from three independent experiments is shown. (F) HEK293T cells coexpressing FLAG-tagged WT TF (WT) or TFdelC (delC) along with MYC-tagged STUB1 were treated with 10 μM MG132 for 16 hours. The lysates were immunoprecipitated with anti-TF antibodies and probed for ubiquitin. The stripped blot was reprobed with FLAG. Five percent of the cell lysates are shown as inputs. Representative immunoblots from three independent experiments are shown. (G) Confocal images of paraffin-embedded sections of an explanted AVF from a 42-year-old male with stage 5 CKD stained with anti-TF and anti-STUB1 antibodies are shown. In different areas of the same AVF, cells with lower STUB1 and higher TF expression are marked by asterisks (*), and cells showing the opposite pattern are marked by crosses (+). The images shown are representative of eight immunofluorescence images acquired from four CKD/ESRD patients. Scale bar, 100 μm. (H) Pipeline of object recognition algorithm developed to correlate cell-level intensity distributions of STUB1 with TF (see Materials and Methods for more details). (I) Eight immunofluorescence images from four explanted AVFs from CKD/ESRD patients were analyzed using an object recognition algorithm. The intensities of TF and STUB1 within image objects containing vSMCs (average of 187 cells per image; total, 1501 cells) were quantified and averaged for each image. Data are shown as means ± SD. (J) Confocal images of paraffin-embedded sections of an explanted AVF from a 47-year-old CKD patient (uremic) and a popliteal artery from a 53-year-old male with normal renal function (nonuremic) (table S1) stained with anti-TF and anti-STUB1 antibodies. Two representatives from a total of eight uremic and nonuremic images are shown. L, lumen; S, subendothelium; M, medium. Asterisks (*) mark areas of cells with low STUB1 and high TF expression similar to those in (G). Scale bars, 100 μm. (K) Confocal images acquired from four explanted AVFs from CKD/ESRD patients and popliteal arteries from patients with normal renal function were analyzed using a pixel-level colocalization algorithm. Percentage colocalization of TF-STUB1 was defined as the fraction of pixels with intensity values greater than image-specific thresholds for STUB1 and TF and was compared between the two groups. Data are shown as means ± SD.

  • Fig. 4. STUB1 modulation regulates TF activity and thrombosis in a postvascular interventional model.

    (A) Scheme of flow-loop preparation. (B) The flow-loop system consists of silastic loops loaded on rotor stages and driven by motors and motion controllers. The tubes injected with the human blood are subjected to coronary-like flow pattern until clotting appears. The wall motion creates bidirectional flows that are measured via onboard, extracorporeal flow probes built into the rotor stages. (C) A representative tube from six independent flow loops seeded with STUB1 WT and KO MEFs is shown (n = 6 per group). (D) Average Hb and LDH from clotted flow loops lined with STUB1 WT and KO MEFs (n = 6 per group). Student’s t test was performed. Data are shown as means ± SEM. (E) Top: Structure of YL-109 (33). Bottom: Primary human aortic vSMCs seeded on fibronectin-coated tubes were stimulated with IS (10 μM) with or without YL-109 (25 μM) for 24 hours before loading. A representative tube from six independent flow loops in each group is shown. (F) Average Hb and LDH from clotted flow loops pretreated with IS with or without YL-109 (n = 6 per group). Data are shown as means ± SEM. (G) Lysates of vSMCs pretreated with 5% uremic serum along with the indicated amount of YL-109 for 24 hours were probed for TF and GAPDH. An equal amount of lysates was probed for STUB1. Representative blots from three independent experiments are shown. Student’s t test was performed, and asterisk (*) indicates significant changes in TF and STUB1 expressions compared to 5% uremic serum (P < 0.05). (H) A serum-starved confluent monolayer of vSMCs was treated with 5% uremic serum and YL-109 at different concentrations, and TF activity was measured in picomolar and normalized to 103 cells. An average of two independent experiments performed in duplicates is shown. A log dose of YL-109 was plotted against TF activity. Data are shown as means ± SD. (I) Primary human aortic vSMCs cotransfected with the FLAG-tag TF and hemagglutinin (HA)–tag ubiquitin plasmids were treated with IS (50 μM) with or without YL-109 (20 μM) for 24 hours and with MG132 (5 μM) for 16 hours before harvest. The lysates were immunoprecipitated using anti-FLAG antibody and probed with anti-HA antibody. Five percent of whole-cell lysates were separately probed for STUB1. A representative blot of three independent experiments is shown.

  • Fig. 5. Perturbations of AHR-STUB1-TF axis normalize the hyperthrombotic uremic phenotype without altering the bleeding risk.

    (A) The experimental design assessing the effect of YL-109 on the IS-specific uremic thrombosis model. A dose of heparin that increases activated partial thromboplastin time (aPTT) to the therapeutic range in mice (35) was administered 2 hours before the procedure. IP, intraperitoneal; PO, per os. (B) Representative traces of carotid artery blood flow showing TtO (arrow) in different groups. (C) An average TtO from each group of animals from three independent experiments is shown. The numbers of animals used include probenecid (n = 9), probenecid + IS (n = 12), probenecid + IS + YL-109 (n = 8), and probenecid + IS + heparin (n = 6). No difference in TtO was observed between probenecid and probenecid + IS + YL-109 (P = 0.65). Heparin significantly prolonged TtO compared to both IS and probenecid control groups (P = 0.021). Data are shown as means ± SD. (D) Mean normalized TF and STUB1 expression in the aortae of five mice injected with IS with or without YL-109 is shown. Student’s t test was performed. Data are shown as means ± SD. (E) Changes in TF expression in the individual aortae of IS + YL-109–treated animals and their respective TtO are shown. A Spearman correlation analysis was performed. (F) IS concentrations in the blood of mice exposed to 0.25% adenine diet for 2 weeks and animals on regular chow (control) (n = 5 per group). Data are shown as means ± SD. (G) An average TtO in control (regular diet) and adenine-induced CKD mice with AHR inhibitor (CH223191) or STUB1 enhancer (YL-109) (n = 5 per group) is shown. #P value compares the adenine and control groups. *P value compares CH223191 and YL-109 groups with the adenine group. Data are shown as means ± SEM. (H) Average tail vein bleeding times for each group (n = 5 per group). Data are shown as means ± SD. (I) Differences in the average tail vein bleeding times (y axis) between probenecid control (non-CKD) and other groups (CKD). The solid line represents the average bleeding time of the control group, and the dotted lines represent two SDs of bleeding time from the control group. The shaded areas are the regions beyond 2 SD.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/417/eaam8475/DC1

    Materials and Methods

    Fig. S1. An indolic solute–specific animal model.

    Fig. S2. STUB1 regulation of TF protein.

    Fig. S3. STUB1 interaction with TF.

    Fig. S4. STUB1 expression in the YL-109–treated flow loops.

    Fig. S5. AHR or STUB1 manipulation in uremic animal model.

    Table S1. Demographic and clinical characteristics of patients included in the study to examine the vascular expression of STUB1 and TF.

  • Supplementary Material for:

    Targeting STUB1–tissue factor axis normalizes hyperthrombotic uremic phenotype without increasing bleeding risk

    Moshe Shashar, Mostafa E. Belghasem, Shinobu Matsuura, Joshua Walker, Sean Richards, Faisal Alousi, Keshab Rijal, Vijaya B. Kolachalama, Mercedes Balcells, Minami Odagi, Kazuo Nagasawa, Joel M. Henderson, Amitabh Gautam, Richard Rushmore, Jean Francis, Daniel Kirchhofer, Kumaran Kolandaivelu, David H. Sherr, Elazer R. Edelman, Katya Ravid, Vipul C. Chitalia*

    *Corresponding author. Email: vichital{at}bu.edu

    Published 22 November 2017, Sci. Transl. Med. 9, eaam8475 (2017)
    DOI: 10.1126/scitranslmed.aam8475

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. An indolic solute–specific animal model.
    • Fig. S2. STUB1 regulation of TF protein.
    • Fig. S3. STUB1 interaction with TF.
    • Fig. S4. STUB1 expression in the YL-109–treated flow loops.
    • Fig. S5. AHR or STUB1 manipulation in uremic animal model.
    • Table S1. Demographic and clinical characteristics of patients included in the study to examine the vascular expression of STUB1 and TF.

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