Journal: Journal of thrombosis and thrombolysis

Article Title: AKT2 regulates endothelial‑mediated coagulation homeostasis and promotes intrathrombotic recanalization and thrombus resolution in a mouse model of venous thrombosis

doi: 10.1007/s11239-020-02112-9

Figure Lengend Snippet: Antibodies used for immunostaining and Western blot experiments

Article Snippet: The positive signal was normalized to the evaluated IVC area, and the mean positive signals were calculated and compared between groups. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Vendor Catalog number Dilution Immunostaining AKT2 Cell Signaling 4691 1:400 p-FOXO1 Thermo Fisher PA5–104977 1:100 Thrombomodulin Santa Cruz SC-6192 1:50 Tissue factor Santa Cruz SC-374441 1:50 Endothelial protein C receptor (EPCR) Santa Cruz SC-28978 1:50 CD31 Santa Cruz SC-376764 1:50 SM22a Abcam AB-89989 1:200 RIP3 Abcam AB-68481 1:200 VEGF Santa Cruz SC-7269 1:50 FSP1 Santa Cruz SC-73355 1:50 SMAD3 Santa Cruz SC-8332 1:50 TGF Santa Cruz SC-146 1:50 Western blot Thrombomodulin Santa Cruz SC-9162 1:200 Tissue factor Santa Cruz SC-65962 1:200 Open in a separate window Antibodies used for immunostaining and Western blot experiments

Techniques: Immunostaining, Western Blot

Journal: Journal of thrombosis and thrombolysis

Article Title: AKT2 regulates endothelial‑mediated coagulation homeostasis and promotes intrathrombotic recanalization and thrombus resolution in a mouse model of venous thrombosis

doi: 10.1007/s11239-020-02112-9

Figure Lengend Snippet: Increased thrombus formation and impaired thrombus resolution in the ligated inferior vena cava (IVC) of Akt2−/− mice. Wild-type (WT) mice and Akt2−/− mice underwent a sham operation or IVC ligation. The thrombus-containing IVC was harvested from each mouse at the designated time. a Schematic illustration of IVC ligation. A midline laparotomy was performed, and the IVC was identified. A 4–0 polypropylene suture was placed longitudinally along the ventral surface of the vein, and the IVC was ligated below the renal veins with a 7–0 polypropylene suture. The 4–0 polypropylene suture was then removed to avoid complete occlusion. Side branches were not ligated. Control mice underwent the same surgical procedure without IVC ligation (sham ligation). b Representative images of immunofluorescence staining and quantification showing that IVC ligation increased AKT2 expression in the IVC of WT mice 1 week after ligation. For the quantification of staining, the mean positive-staining area was normalized with the evaluated area. All values represent the mean (n = 6 per group). P-values were obtained by performing the Welch’s t test. c Time-course analysis showing that the mean weight of the combined IVC and thrombus was significantly greater in Akt2−/− mice than in WT mice from day 7 to day 28 after ligation. Furthermore, the mean weight of the combined IVC and thrombus decreased more slowly in Akt2−/− mice (significantly reduced at day 21 after ligation) than in WT mice (significantly reduced at day 14 after ligation). All values represent the mean (n = 6 per group). P-values were obtained by performing two-way ANOVA analysis followed by post-hoc pairwise comparisons with the Bonferroni correction. d Representative hematoxylin and eosin staining showing larger thrombi in the IVC of Akt2−/− mice than in that of WT mice at different times after ligation

Article Snippet: The positive signal was normalized to the evaluated IVC area, and the mean positive signals were calculated and compared between groups. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Vendor Catalog number Dilution Immunostaining AKT2 Cell Signaling 4691 1:400 p-FOXO1 Thermo Fisher PA5–104977 1:100 Thrombomodulin Santa Cruz SC-6192 1:50 Tissue factor Santa Cruz SC-374441 1:50 Endothelial protein C receptor (EPCR) Santa Cruz SC-28978 1:50 CD31 Santa Cruz SC-376764 1:50 SM22a Abcam AB-89989 1:200 RIP3 Abcam AB-68481 1:200 VEGF Santa Cruz SC-7269 1:50 FSP1 Santa Cruz SC-73355 1:50 SMAD3 Santa Cruz SC-8332 1:50 TGF Santa Cruz SC-146 1:50 Western blot Thrombomodulin Santa Cruz SC-9162 1:200 Tissue factor Santa Cruz SC-65962 1:200 Open in a separate window Antibodies used for immunostaining and Western blot experiments

Techniques: Ligation, Control, Immunofluorescence, Staining, Expressing

Journal: Journal of thrombosis and thrombolysis

Article Title: AKT2 regulates endothelial‑mediated coagulation homeostasis and promotes intrathrombotic recanalization and thrombus resolution in a mouse model of venous thrombosis

doi: 10.1007/s11239-020-02112-9

Figure Lengend Snippet: Significantly increased tissue factor (TF) expression and decreased thrombomodulin (TM) expression in the ligated inferior vena cava (IVC) of Akt2−/− mice. Wild-type (WT) mice and Akt2−/− mice underwent a sham operation or IVC ligation. The thrombus-containing IVC was harvested from each mouse 2 weeks after ligation. Representative images of immunofluorescence staining and quantification showing increased TF expression (a) and decreased TM expression (b) in the ligated IVC of Akt2−/− mice. For the quantification of staining, the mean positive-staining area was normalized with the evaluated area. All values represent the mean (n = 6 per group). P-values were obtained by performing multiple comparisons in one-way ANOVA analysis followed by the Tukey multiple comparisons test. c Representative images of immunofluorescence staining showing decreased endothelial protein C receptor (EPCR) expression in the ligated IVC of Akt2−/− mice

Article Snippet: The positive signal was normalized to the evaluated IVC area, and the mean positive signals were calculated and compared between groups. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Vendor Catalog number Dilution Immunostaining AKT2 Cell Signaling 4691 1:400 p-FOXO1 Thermo Fisher PA5–104977 1:100 Thrombomodulin Santa Cruz SC-6192 1:50 Tissue factor Santa Cruz SC-374441 1:50 Endothelial protein C receptor (EPCR) Santa Cruz SC-28978 1:50 CD31 Santa Cruz SC-376764 1:50 SM22a Abcam AB-89989 1:200 RIP3 Abcam AB-68481 1:200 VEGF Santa Cruz SC-7269 1:50 FSP1 Santa Cruz SC-73355 1:50 SMAD3 Santa Cruz SC-8332 1:50 TGF Santa Cruz SC-146 1:50 Western blot Thrombomodulin Santa Cruz SC-9162 1:200 Tissue factor Santa Cruz SC-65962 1:200 Open in a separate window Antibodies used for immunostaining and Western blot experiments

Techniques: Expressing, Ligation, Immunofluorescence, Staining

Journal: Journal of thrombosis and thrombolysis

Article Title: AKT2 regulates endothelial‑mediated coagulation homeostasis and promotes intrathrombotic recanalization and thrombus resolution in a mouse model of venous thrombosis

doi: 10.1007/s11239-020-02112-9

Figure Lengend Snippet: Significantly increased cell death in the ligated inferior vena cava (IVC) of Akt2−/− mice. Wild-type (WT) mice and Akt2−/− mice underwent a sham operation or IVC ligation. The thrombus-containing IVC was harvested at 2 weeks after ligation. a Representative images of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and quantification showing that apoptosis was significantly increased in the lesion areas of the ligated IVC of Akt2−/− mice, particularly in thrombus areas. For the quantification of staining, the number of TUNEL-positive cells was normalized to the total number of cells evaluated. All values represent the mean (n = 6 in each group). P-values were obtained by performing one-way ANOVA analysis followed by the Holm-Sidak multiple comparisons test. b Representative images of immunofluorescence staining and quantification showing a significant increase in the levels of the necroptosis molecule receptor-interacting protein kinase 3 (RIP3) in the ligated IVC of Akt2−/− mice. For the quantification of staining, the mean positive-staining area was normalized with the evaluated aortic area. All values represent the mean (n = 6 per group). P-values were obtained by performing one-way ANOVA analysis followed by the Tukey multiple comparisons test

Article Snippet: The positive signal was normalized to the evaluated IVC area, and the mean positive signals were calculated and compared between groups. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Vendor Catalog number Dilution Immunostaining AKT2 Cell Signaling 4691 1:400 p-FOXO1 Thermo Fisher PA5–104977 1:100 Thrombomodulin Santa Cruz SC-6192 1:50 Tissue factor Santa Cruz SC-374441 1:50 Endothelial protein C receptor (EPCR) Santa Cruz SC-28978 1:50 CD31 Santa Cruz SC-376764 1:50 SM22a Abcam AB-89989 1:200 RIP3 Abcam AB-68481 1:200 VEGF Santa Cruz SC-7269 1:50 FSP1 Santa Cruz SC-73355 1:50 SMAD3 Santa Cruz SC-8332 1:50 TGF Santa Cruz SC-146 1:50 Western blot Thrombomodulin Santa Cruz SC-9162 1:200 Tissue factor Santa Cruz SC-65962 1:200 Open in a separate window Antibodies used for immunostaining and Western blot experiments

Techniques: Ligation, TUNEL Assay, Staining, Immunofluorescence

Journal: Journal of thrombosis and thrombolysis

Article Title: AKT2 regulates endothelial‑mediated coagulation homeostasis and promotes intrathrombotic recanalization and thrombus resolution in a mouse model of venous thrombosis

doi: 10.1007/s11239-020-02112-9

Figure Lengend Snippet: Impaired intrathrombotic angiogenesis and recanalization and increased fibrotic remodeling in the ligated inferior vena cava (IVC) of Akt2−/− mice. Wild-type (WT) mice and Akt2−/− mice underwent a sham operation or IVC ligation. The thrombus-containing IVC was harvested at 2 weeks after ligation. a The IVC from WT or Akt2–/– mice was stained with endothelial marker CD31. Intrathrombotic CD31-positive neovessels with tube-like formation were counted in 5 high-magnification fields (× 400). Representative images of staining and quantification of the number of neovessels showing a significantly decreased number of neovessels in the ligated IVC of Akt2−/− mice. All values represent the mean (n = 6 per group). P-values were obtained by performing one-way ANOVA analysis followed by the Tukey multiple comparisons test. b Representative images of immunofluorescence staining and quantification showing decreased vascular endothelial growth factor (VEGF) expression in the ligated IVC of Akt2−/− mice. For the quantification of staining, the mean positive-staining area was normalized with the evaluated IVC area. All values represent the mean (n = 6 per group). P-values were obtained by performing multiple comparisons in one-way ANOVA analysis followed by the Tukey multiple comparisons test. c Representative images of immunofluorescence staining showing fibroblast specific protein (FSP)-1–positive cells in the ligated IVC of WT mice, which were even more abundant in the ligated IVC of Akt2−/− mice. d Representative images of double immunofluorescence staining showing the colocalization of FSP-1 with the smooth muscle cell marker SM22-α in the vein wall and in the thrombus of ligated IVC in Akt2−/− mice. e, f Representative images of immunofluorescence staining showing increased levels of e SMAD3 and f TGF-β in the ligated IVC of Akt2−/− mice compared with WT mice

Article Snippet: The positive signal was normalized to the evaluated IVC area, and the mean positive signals were calculated and compared between groups. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Vendor Catalog number Dilution Immunostaining AKT2 Cell Signaling 4691 1:400 p-FOXO1 Thermo Fisher PA5–104977 1:100 Thrombomodulin Santa Cruz SC-6192 1:50 Tissue factor Santa Cruz SC-374441 1:50 Endothelial protein C receptor (EPCR) Santa Cruz SC-28978 1:50 CD31 Santa Cruz SC-376764 1:50 SM22a Abcam AB-89989 1:200 RIP3 Abcam AB-68481 1:200 VEGF Santa Cruz SC-7269 1:50 FSP1 Santa Cruz SC-73355 1:50 SMAD3 Santa Cruz SC-8332 1:50 TGF Santa Cruz SC-146 1:50 Western blot Thrombomodulin Santa Cruz SC-9162 1:200 Tissue factor Santa Cruz SC-65962 1:200 Open in a separate window Antibodies used for immunostaining and Western blot experiments

Techniques: Ligation, Staining, Marker, Immunofluorescence, Expressing, Double Immunofluorescence Staining

Journal: Journal of thrombosis and thrombolysis

Article Title: AKT2 regulates endothelial‑mediated coagulation homeostasis and promotes intrathrombotic recanalization and thrombus resolution in a mouse model of venous thrombosis

doi: 10.1007/s11239-020-02112-9

Figure Lengend Snippet: Inhibition of the FOXO1-mediated regulation of thrombomodulin (TM) and tissue factor (TF) expression by AKT2. Human umbilical vein endothelial cells (HUVEC) were transfected with wild-type (WT)-AKT2 or AKT2 siRNA. Western blot analysis a and results of quantitative RT-PCR b showing a trend of induced TM expression and reduced TF expression by AKT2 overexpression. mRNA levels were normalized with those of β-actin and are expressed as the percentage of the control. Data represent the mean of 3 biologic repeats. c HUVECs were transfected with plasmids expressing WT-FOXO1, constitutively active (CA)-FOXO1, or dominant negative (DN)-FOXO1. Quantitative RT-PCR results showing that WT-FOXO1 and CA-FOXO1 induced a trend of reduced TM expression and induced TF expression. DN-FOXO1 had opposite effects. mRNA levels were normalized with those of β-actin and are expressed as the percentage of the control. Data represent the mean of 3 biologic repeats. d HUVECs were transfected with WT-FOXO1 plasmid in the presence of WT-AKT2 and AKT2 siRNA. Quantitative RT-PCR results showing that AKT2 reversed the FOXO1-mediated inhibition of TM and stimulation of TF. The relative levels of mRNA are expressed as the percentage of the control. Data represent the mean of > 3 biologic repeats. P-values were obtained by performing multiple comparisons in one-way ANOVA analysis followed by the Tukey multiple comparisons test. e Wild-type (WT) mice and Akt2−/− mice underwent a sham operation or IVC ligation. The thrombus-containing IVC was harvested at 2 weeks after ligation. Representative images of immunofluorescence staining showing increased phospho FOXO1 (p-FOXO1) levels in the ligated IVC of Akt2−/− mice compared with WT mice

Article Snippet: The positive signal was normalized to the evaluated IVC area, and the mean positive signals were calculated and compared between groups. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Vendor Catalog number Dilution Immunostaining AKT2 Cell Signaling 4691 1:400 p-FOXO1 Thermo Fisher PA5–104977 1:100 Thrombomodulin Santa Cruz SC-6192 1:50 Tissue factor Santa Cruz SC-374441 1:50 Endothelial protein C receptor (EPCR) Santa Cruz SC-28978 1:50 CD31 Santa Cruz SC-376764 1:50 SM22a Abcam AB-89989 1:200 RIP3 Abcam AB-68481 1:200 VEGF Santa Cruz SC-7269 1:50 FSP1 Santa Cruz SC-73355 1:50 SMAD3 Santa Cruz SC-8332 1:50 TGF Santa Cruz SC-146 1:50 Western blot Thrombomodulin Santa Cruz SC-9162 1:200 Tissue factor Santa Cruz SC-65962 1:200 Open in a separate window Antibodies used for immunostaining and Western blot experiments

Techniques: Inhibition, Expressing, Transfection, Western Blot, Quantitative RT-PCR, Over Expression, Control, Dominant Negative Mutation, Plasmid Preparation, Ligation, Immunofluorescence, Staining