Journal: bioRxiv

Article Title: Ubiquitin ligase CHFR impairs Tie2 signaling via K 48 -linked ubiquitylation and degradation of Akt1 in endothelial cells

doi: 10.64898/2026.03.31.715582

Figure Lengend Snippet: a HMEC were transfected with scrambled-siRNA (sc-siRNA) or CHFR-siRNA. At 72 h post-transfection, cells stimulated with rAng1 (400 ng/ml) for different time intervals were used for IB analysis to determine phosphorylation of Akt1 at S473 and T308 ( n = 3 independent experiments). b Control HMEC and CHFR knockdown HMEC stimulated with rAng1 as above were stained with phospho-473-Akt1 specific antibody. c Control HMEC and CHFR knockdown HMEC were transfected with the Akt1 biosensor plasmid and then stimulated with rAng1 as above to assess live cell Akt1 activity by measuring the FRET ratio ( p <0.0001) ( left panel shows basal Akt activity; right panel shows Akt activity in response to rAng1 challenge). d-f Control HMEC and CHFR-siRNA treated HMEC stimulated with rAng1 were used for IB analysis to determine tyrosine phosphorylation Tie2 ( d ), phosphorylation of GSK3β at S9 and phosphorylation of β-catenin ( e ), ( n = 3 independent experiments). a, d-e Shown are mean values ± SEM ( n = 3 independent experiments; two-way ANOVA followed by Tukey’s post-hoc test).

Article Snippet: Rabbit polyclonal antibody (pAb) against phospho-Tie2 (catalog #AF3909; IB, 1:1000), goat polyclonal antibody (pAb) against PDGFRβ (catalog #AF1042; IS, 1:100) were from R&D system.

Techniques: Transfection, Phospho-proteomics, Control, Knockdown, Staining, Plasmid Preparation, Activity Assay

Journal: bioRxiv

Article Title: Ubiquitin ligase CHFR impairs Tie2 signaling via K 48 -linked ubiquitylation and degradation of Akt1 in endothelial cells

doi: 10.64898/2026.03.31.715582

Figure Lengend Snippet: a HMEC were transfected with WT or K/R-mutant Akt1 constructs. At 36 h after transfection, cells were treated with LPS (5 μg/ml) for 0, 12, and 24h and then cells were used for IB to determine expression of Akt1 and VE-cadherin. Shown are mean values ± SEM (n = 3 independent experiments; two-way ANOVA followed by Tukey’s post hoc test). b TIME endothelial cells (telomerase-immortalized human dermal microvascular endothelial cell line) were transfected with WT or K/R mutant Akt1 constructs and stimulated with LPS (5 μg/ml) for 0 and 6 h. Confocal imaging showed that expression of K/R mutant Akt1 prevents degradation of VE-cadherin. c WT mice were injected (i.v.) with liposome-encapsulated pmCherry-tagged WT or K/R mutant Akt1 constructs. Lungs harvested 96 h after injection were subjected to cryosection and stained with EC marker antibody vWF (green). Confocal imaging confirms expression of pmCherry-Akt1 (red) plasmid in lung endothelial cells. d-f Liposome-mediated delivery of Akt1 (WT) or K/R-mutated Akt1 in WT mice prevents degradation of VE-cadherin, mitigates LPS-induced lung vascular leak (EBA uptake), and reduces PMN transmigration (MPO assay). Shown are mean values ± SEM ( n = 3 or n = 5 mice/group; two-way ANOVA followed by Tukey’s post hoc test). g Model for E3 ligase CHFR regulation of endothelial junctional barrier integrity. Under baseline condition, constitutive Ang1-Tie2 signaling in EC maintains endothelial junctional barrier through Akt1 activation-mediated inhibition of the transcription factor FoxO1 activation and Ang-2 expression. During vascular inflammatory conditions such as sepsis, TLR4 signaling induces the expression of E3 ligase CHFR in a FoxO1-dependent manner. Then the upregulated CHFR mediates K 48 -linked polyubiquitylation and degradation of Akt1 and VE-cadherin (Tiruppathi et al., 2023) to disassemble EC junctional barrier. CHFR-mediated loss of FoxO1 negative regulator Akt1 expression in EC leads to increased FoxO1 expression which in turn promotes sustained expression of Ang-2 in EC to induce life-threatening pulmonary edema.

Article Snippet: Rabbit polyclonal antibody (pAb) against phospho-Tie2 (catalog #AF3909; IB, 1:1000), goat polyclonal antibody (pAb) against PDGFRβ (catalog #AF1042; IS, 1:100) were from R&D system.

Techniques: Transfection, Mutagenesis, Construct, Expressing, Imaging, Injection, Staining, Marker, Plasmid Preparation, Transmigration Assay, MPO Assay, Activation Assay, Inhibition

Journal: The Journal of Clinical Investigation

Article Title: TIE2 activation by antibody-clustered endogenous angiopoietin-2 prevents capillary loss and fibrosis in experimental kidney disease

doi: 10.1172/JCI190286

Figure Lengend Snippet: ( A ) Schematic diagram of the experimental setup with UUO, contralateral control kidney (CL), and ABTAA (or IgG) administration. ( B and C ) Schematic diagram of ABTAA function ( B ) and inducible conditional mouse lines used in the study ( C ). TAM, tamoxifen induction. ( D and E ) pTIE2/TIE2 in 3-day UUO kidneys in ABTAA (ABT) and Veptp iECKO (Ve KO ) mice, respectively. Data are based on n = 3–10 mice/group and 3 ( D ) and 1 ( E ) blots (see ). ( F ) VEPTP in kidney lysates from CL and 3-day UUO kidneys in WT and Veptp iECKO mice. Data are based on n = 3 mice/group and 1 blot (see ). ( G ) TIE2 protein measured by ELISA in kidney homogenates from CL and 3-day UUO kidneys in Tie2 iECKO (T2 KO ) mice. Data are based on n = 3–5 mice/group. ( H ) Renal perfusion measured with ultrasound contrast imaging in 2-day UUO/CL kidneys from ABTAA-treated, Veptp iECKO , Tie2 iECKO , and Pdgfb iKO (Pb KO ) mice and their respective controls. The same group of sham-operated mice was used to establish baseline perfusion levels for comparison with UUO-injured kidneys. Data are based on n = 5–8 mice/group. ( I – K ) Immunohistochemistry and quantification from renal cortex for endothelial markers (endomucin [EMCN] and podocalyxin [PODXL]) in 3-day UUO kidneys from ABTAA-treated, Veptp iECKO , Tie2 iECKO , and Pdgfb iKO mice and their controls. Data are based on n = 5–11 mice/group and >600 images/marker. Scale bars: 50 μm. Data represent mean ± SD, and each symbol represents 1 mouse (females, magenta; males, cyan). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001, determined by 1-way ANOVA and Tukey’s post hoc test ( F – H , J , and K ) or unpaired 2-tailed Student’s t test ( D and E ).

Article Snippet: The membranes were stripped and reprobed with goat anti-mouse TIE2 Ab (AF762; R&D Systems).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Imaging, Comparison, Immunohistochemistry, Marker

Journal: The Journal of Clinical Investigation

Article Title: TIE2 activation by antibody-clustered endogenous angiopoietin-2 prevents capillary loss and fibrosis in experimental kidney disease

doi: 10.1172/JCI190286

Figure Lengend Snippet: ( A and B ) Semiquantitative grading of capillary length with fenestrations from micrographs of peritubular capillaries in 3-day UUO kidneys from ABTAA-treated (ABT), Veptp iECKO (Ve KO ), and Tie2 iECKO (T2 KO ) mice. Data are based on n = 4–5 mice/group and scoring of 892 micrographs. Scoring is based on percentage of endothelium with fenestrations as follows: 0, 0%–5%; 1, 6%–25%; 2, 26%–50%; 3, 51%–75%; and 4, 76%–100%. The fenestrated endothelium is indicated by an arrow. Scale bars: 2 μm. ( C ) Quantification of endothelial nuclei in Tie2 iECKO and WT mice from 1-, 3-, and 10-day UUO kidneys. Data are based on n = 4–7 mice/group, and 310 images were quantified. ( D ) Representative image with immunohistochemistry for endomucin (cyan) together with the endothelial Cdh5 -Tdtomato lineage tracer (magenta). Scale bars: 50 μm. Data represent mean ± SD, and each symbol represents 1 mouse (females, magenta; males, cyan). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001, determined by 1-way ANOVA and Tukey’s post hoc test ( B and C ).

Article Snippet: The membranes were stripped and reprobed with goat anti-mouse TIE2 Ab (AF762; R&D Systems).

Techniques: Immunohistochemistry

Journal: The Journal of Clinical Investigation

Article Title: TIE2 activation by antibody-clustered endogenous angiopoietin-2 prevents capillary loss and fibrosis in experimental kidney disease

doi: 10.1172/JCI190286

Figure Lengend Snippet: ( A – C ) Immunohistochemistry and quantifications from renal cortex for fibrosis markers (aSMA and vimentin [VIM]) in 3-day UUO kidneys from ABTAA-treated (ABT), Veptp iECKO (Ve KO ), Tie2 iECKO (T2 KO ), and Pdgfb iKO (Pb KO ) mice and their controls. Data are based on n = 5–11 mice/group and quantifications from >600 images/marker. Scale bars: 50 μm. ( D ) Renal Col1a1 expression in ABTAA-treated mice. Data are based on n = 6 mice/group. ( E ) Renal Pdgfrb expression in Pdgfb iKO mice. Data are based on n = 5–6 mice/group. ( F ) Gene expression of Col1a1 , Tagln , and Fn1 in 3-day UUO kidneys from Tie2 iECKO mice. Data are based in n = 5–6 mice/group. Data represent mean ± SD, and each symbol represents 1 mouse (females, magenta; males, cyan). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001, determined by 1-way ANOVA and Tukey’s post hoc test ( B – F ).

Article Snippet: The membranes were stripped and reprobed with goat anti-mouse TIE2 Ab (AF762; R&D Systems).

Techniques: Immunohistochemistry, Marker, Expressing, Gene Expression

Journal: The Journal of Clinical Investigation

Article Title: TIE2 activation by antibody-clustered endogenous angiopoietin-2 prevents capillary loss and fibrosis in experimental kidney disease

doi: 10.1172/JCI190286

Figure Lengend Snippet: ( A ) Breeding strategy for Tie2 iECKO mice with endothelial lineage tracer and myofibroblast reporter. ( B and C ) Imaging and quantification of Pdgfra -H2BGFP + nuclei (cyan) in renal cortex of 3- and 10-day UUO kidneys in WT and Tie2 iECKO (T2 KO ) mice. Data are based on n = 3–6 mice/group and quantifications from >180 images. Scale bars: 50 μm. ( D ) Representative confocal image stack (30 μm) from renal cortex showing Cdh5 -TdTomato (magenta) and Pdgfra -H2BGFP (cyan) in renal cortex 10 days after UUO in a Tie2 iECKO mouse. Scale bar: 10 μm. ( E ) Confocal images from renal cortex showing tubular epithelial marker NaK/ATPase (blue), Pdgfra -GFP (cyan), and Cdh5 -TdTomato (magenta) 10 days after UUO in WT and Tie2 iECKO mice. Representative images of n = 3 mice per group. Scale bars: 25 μm. Data in graphs represent mean ± SD, and each symbol represents 1 mouse (females, magenta; males, cyan). ** P < 0.01, *** P < 0.001, and **** P < 0.0001, determined by 1-way ANOVA and Tukey’s post hoc test ( C ).

Article Snippet: The membranes were stripped and reprobed with goat anti-mouse TIE2 Ab (AF762; R&D Systems).

Techniques: Imaging, Marker

Journal: The Journal of Clinical Investigation

Article Title: TIE2 activation by antibody-clustered endogenous angiopoietin-2 prevents capillary loss and fibrosis in experimental kidney disease

doi: 10.1172/JCI190286

Figure Lengend Snippet: ( A ) Tubular segments with pathological vacuoles (red arrow) in kidney sections stained with toluidine blue. ( B ) Quantification of tubular segments with vacuoles from ABTAA-treated (ABT), Veptp iECKO (Ve KO ), and Tie2 iECKO (T2 KO ) mice. Data are based on n = 4–5 mice/group and >10,000 tubular cross sections. ( C ) RNA-ISH for Pecam1 (cyan) and Pdgfb (magenta) in 3-day UUO kidneys. Representative image of n = 4 mice. Scale bars: 50 μm. ( D and E ) Gene expression of Kim1 , Pdgfb , Pdgfrb , Tie2 , and Angpt1 in UUO kidneys from indicated time points. Data are based on n = 3–7 mice/group. ( F ) RNA-ISH for Angpt1 (magenta), mesenchymal marker Pdgfrb (cyan), and tubular marker Atp1a1 (blue) in 3-day UUO kidneys. Representative image of n = 3 mice. Scale bars: 50 μm. ( G – I ) Expression of Pdgfb/PDGFB in 3-day UUO kidneys from ABTAA-treated, Veptp iECKO , and Pdgfb iKO (Pb KO ) mice. Data are based on n = 5–9 mice/group. ( J ) Patient data retrieved from Nephroseq for renal CDH5 (endothelial marker), ANGPT2 , PDGFB , and ANGPT1 expression in CKD and renal dysfunction compared with normal human kidney. Data in graphs ( B , D , E , and G – I ) represent mean ± SD, and each symbol represents 1 mouse (females, magenta; males, cyan). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001, determined by 1-way ANOVA. Human data ( J ) represent Log2 expression and statistical differences from Nephroseq (see Methods).

Article Snippet: The membranes were stripped and reprobed with goat anti-mouse TIE2 Ab (AF762; R&D Systems).

Techniques: Staining, Gene Expression, Marker, Expressing

Journal: The Journal of Clinical Investigation

Article Title: TIE2 activation by antibody-clustered endogenous angiopoietin-2 prevents capillary loss and fibrosis in experimental kidney disease

doi: 10.1172/JCI190286

Figure Lengend Snippet: ( A ) Schematic diagram of administration of ABTAA for evaluation in 10-day UUO kidneys. ( B – F ) Immunohistochemistry of renal cortex for capillary density (endomucin [EMCN] and podocalyxin [PODXL]) and tubulointerstitial fibrosis (aSMA and vimentin) in 10-day UUO kidneys from ABTAA-treated mice (ABT) and Tie2 iECKO (T2 KO ) mice. Data are based on n = 6–7 mice/group and quantification of >220 images/marker. Scale bars: 50 μm. ( E ) Protein concentration for PDGFB in 10-day UUO kidneys from ABTAA-treated mice. Data are based on n = 5 mice/group. Data in graphs represent mean ± SD, and each symbol represents 1 mouse (females, magenta; males, cyan). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001, determined by 1-way ANOVA and Tukey’s post hoc test.

Article Snippet: The membranes were stripped and reprobed with goat anti-mouse TIE2 Ab (AF762; R&D Systems).

Techniques: Immunohistochemistry, Marker, Protein Concentration