Journal: bioRxiv

Article Title: Thrombospondin-1 inhibits alternative complement pathway activation in vasculitis synergistically to factor H

doi: 10.1101/2024.07.31.606000

Figure Lengend Snippet: (A) TSP-1 inhibits AP activation in contrast to TSP-5. AP in NHS was activated on LPS coated wells and with increasing concentrations of FH, Eculizumab, TSP-1 or TSP-5. Platelet derived TSP-1 (p-TSP-1) was used as an additional control to exclude artifacts caused by the histidine tag used for purification. (B) TSP-1 protects sheep erythrocytes from AP mediated lysis in the absence of FH. Sheep erythrocytes were incubated with FH depleted serum and increasing concentrations of FH, TSP-1 or TSP-5. Results are expressed as means ± SD. AP and hemolytic activity were normalized against untreated control samples. Data was fitted using nonlinear regression. (C) TSP-1 binds to central proteins of the alternative pathway. Complement proteins FH, FB, C3, C5, C6, C7, C8, C9 or BSA were coated on microtiter plates and incubated with recombinant TSP-1. Bound TSP-1 was determined using specific antibodies. (D) Surface Plasmon Resonance (SPR) Biacore measurements demonstrating the binding of TSP-1 to key proteins of the alternative complement pathway. TSP-1 was immobilized on CM5 chips at a concentration of 0.1 µM. Binding interactions with complement proteins FH, FB, C3, C3b, C5, and C8 were assessed at various concentrations (12.3, 37.03, 111.1, 333.3, 1000 nM). The binding data were fitted using a 1:1 Langmuir binding model to determine on and off rates, which were then used to calculate affinity constants (Kd). The graph depicts a summary of the binding of complement proteins to TSP-1 at increasing concentrations. Average Kd values, calculated from three repeated measurements, are presented in the accompanying table. Data is represented as mean ± SD

Article Snippet: Human TSP-1 cDNA was obtained from Sinobiological (HG10508-UT) and subcloned into a pFastbac1 vector containing a gp67 signal peptide for secretion and an N-terminal 10xHis tag for purification.

Techniques: Activation Assay, Derivative Assay, Control, Purification, Lysis, Incubation, Activity Assay, Recombinant, SPR Assay, Binding Assay, Concentration Assay

Journal: bioRxiv

Article Title: Thrombospondin-1 inhibits alternative complement pathway activation in vasculitis synergistically to factor H

doi: 10.1101/2024.07.31.606000

Figure Lengend Snippet: TSP-1 modulates complement at the C3 level of the complement cascade. (A) TSP-1 protects FB from cleavage by FD. FB was incubated with FD, C3b, and varying concentrations of TSP-1. Subsequently, FB and its cleavage products were visualized by coomassie staining. Graph on the right illustrates band intensity of FB and its cleavage products. (B) TSP-1 inhibits cleavage of C3 by the AP C3 convertase. C3 convertase was generated by incubating C3(H2O) with FB and FD, followed by the addition of C3 in the presence or absence of TSP-1 or FH. As a positive control, CVF C3 convertase (CVF + FB + FD) was utilized. The graph on the right depicts the band intensity of C3α‘ chain. Results are shown as means.

Article Snippet: Human TSP-1 cDNA was obtained from Sinobiological (HG10508-UT) and subcloned into a pFastbac1 vector containing a gp67 signal peptide for secretion and an N-terminal 10xHis tag for purification.

Techniques: Incubation, Staining, Generated, Positive Control

Journal: bioRxiv

Article Title: Thrombospondin-1 inhibits alternative complement pathway activation in vasculitis synergistically to factor H

doi: 10.1101/2024.07.31.606000

Figure Lengend Snippet: TSP-1 modulates complement at the C5 level of the complement cascade. (A) TSP-1 inhibits cleavage of C5. Cobra venom factor (CVF) convertase (CVFBb) was generated and added to C5 preincubated with FH, Eculizumab, MFHR1 or TSP-1. The amount of released C5a was quantified by ELISA. Results are shown as mean ± SD. *P ≤ 0.05, **P ≤ 0.01, ANOVA using Dunnett’s multiple comparison test. (B) TSP-1 inhibits the formation of the MAC. Sheep Erythrocytes were premixed with C7 (9 nM), C8 (7 nM) and C9 (15 nM). BSA, Eculizumab, MFHR1 or TSP-1 (1.3 µM each) were preincubated with C5b-6 (0.7 nM) and then added to the erythrocytes. Bars represent means ± SD of 3 independent experiments, **P ≤ 0.01, ***P ≤ 0.01, One-way ANOVA with Tukey’s post hoc test for comparison against control.

Article Snippet: Human TSP-1 cDNA was obtained from Sinobiological (HG10508-UT) and subcloned into a pFastbac1 vector containing a gp67 signal peptide for secretion and an N-terminal 10xHis tag for purification.

Techniques: Combined Bisulfite Restriction Analysis Assay, Generated, Enzyme-linked Immunosorbent Assay, Comparison, Control

Journal: bioRxiv

Article Title: Thrombospondin-1 inhibits alternative complement pathway activation in vasculitis synergistically to factor H

doi: 10.1101/2024.07.31.606000

Figure Lengend Snippet: (A) TSP-1 prevents C3 deposition on PNH erythrocytes. PNH Erythrocytes were incubated with acidified serum alone, Eculizumab or with a combination of Eculizumab and TSP-1 or the C3 inhibitor Pegcetacoplan. Erythrocytes were stained for CD59 and C3 and percentages of positive and negative stained cells analyzed by flow cytometry. Eculizumab prevents lysis of PNH erythrocytes but leaves C3 depositions on the surface of CD59 negative erythrocytes. Combination of Eculizumab with TSP-1 or Pegcetacoplan prevents C3 deposition on CD59 negative PNH erythrocytes. (B) Analysis of C3 deposition on PNH erythrocytes from three different patients’ samples. Combined treatment of erythrocytes with Eculizumab and TSP-1 or Eculizumab and Pegcetacoplan significantly reduced the amount of C3 positive CD59 negative cells compared to Eculizumab treatment alone. Bars represent means ± SD of 3 independent experiments, **P ≤ 0.01, One-way ANOVA with Tukey’s post hoc test. aNHS – acidified normal human serum.

Article Snippet: Human TSP-1 cDNA was obtained from Sinobiological (HG10508-UT) and subcloned into a pFastbac1 vector containing a gp67 signal peptide for secretion and an N-terminal 10xHis tag for purification.

Techniques: Incubation, Staining, Flow Cytometry, Lysis

Journal: bioRxiv

Article Title: Thrombospondin-1 inhibits alternative complement pathway activation in vasculitis synergistically to factor H

doi: 10.1101/2024.07.31.606000

Figure Lengend Snippet: TSP-1 inhibits hemolytic activity and pathogenic C3 deposition on endothelial cells when added to the serum of aHUS patients. (A) TSP-1 protects sheep erythrocytes from complement induced lysis in aHUS1 serum. Sheep erythrocytes were incubated with aHUS1 serum and increasing concentrations of FH, TSP-1 or TSP-5. Data was normalized against erythrocytes treated with aHUS1 serum without inhibitors. Bars represent means ± SD of 3 independent experiments. (B) Representative fluorescence images of C3 deposits on HMEC-1 cells treated with aHUS sera. HMEC-1 cells were activated with ADP and incubated with 50% normal human serum or aHUS serum with or without 1µM TSP-1 or FH and stained for C3 deposits. (C) TSP-1 prevents C3 deposition on endothelial cells treated with aHUS sera. Mean fluorescence analysis shows that aHUS2 and aHUS 3 serum causes strong deposition of C3 molecules on HMEC-1, which could be prevented by addition of either FH or TSP-1 into the serum. C3 fluorescence intensity was measured in at least 5 randomly chosen high power fields. Results are shown as mean ± SD. ***P ≤ 0.01, One-way ANOVA using Turkey’s multiple comparison test. Scale Bar = 100 µl

Article Snippet: Human TSP-1 cDNA was obtained from Sinobiological (HG10508-UT) and subcloned into a pFastbac1 vector containing a gp67 signal peptide for secretion and an N-terminal 10xHis tag for purification.

Techniques: Activity Assay, Lysis, Incubation, Fluorescence, Staining, Comparison

Journal: bioRxiv

Article Title: Thrombospondin-1 inhibits alternative complement pathway activation in vasculitis synergistically to factor H

doi: 10.1101/2024.07.31.606000

Figure Lengend Snippet: Potential role of TSP-1 in regulating complement activity on endothelial cells. Knockdown of TSP-1 increases C3 deposition on HUVECs. Left side: Representative fluorescence images of siRNA-treated HUVECs. Cells were transfected with control siRNA or TSP-1 siRNA and stimulated with histamine to mimic inflammation. In parallel, recombinant TSP-1 (rec. TSP-1) was supplemented into TSP-1 siRNA-treated cell supernatants. After 45 minutes of incubation, cells were fixed and stained for DAPI (blue), C3 (green), or TSP-1 (red). Right side: Analysis of TSP-1 and C3 staining. TSP-1 siRNA-treated samples exhibited a significant decrease in TSP-1 staining, which was reinstated by the addition of TSP-1 to the supernatant. In parallel, TSP-1 knockdown led to a substantial increase in C3 deposits on HUVECs, and this effect was ameliorated by supplementing the supernatant with TSP-1. Fluorescence intensity was measured in 5 randomly chosen high-power fields. Results are shown as mean ± SD. *P≤ 0.05, **P ≤ 0.01, One-way ANOVA using Turkey’s multiple comparison test. Scale Bar = 100 µm.

Article Snippet: Human TSP-1 cDNA was obtained from Sinobiological (HG10508-UT) and subcloned into a pFastbac1 vector containing a gp67 signal peptide for secretion and an N-terminal 10xHis tag for purification.

Techniques: Activity Assay, Knockdown, Fluorescence, Transfection, Control, Recombinant, Incubation, Staining, Comparison

Journal: bioRxiv

Article Title: Thrombospondin-1 inhibits alternative complement pathway activation in vasculitis synergistically to factor H

doi: 10.1101/2024.07.31.606000

Figure Lengend Snippet: Potential role of TSP-1 in SARS-CoV-2 endothelial inflammation. SARS-CoV-2 mediated inflammation demonstrates the interplay between TSP-1, vWF and the complement system on endothelial cells. HUVECs were stimulated with hirudin treated blood, in the presence or absence of 5 µg of SARS-CoV-2 S1 protein, for 45 minutes. Following fixation, immunostaining was performed to visualize TSP-1 (red), C3 (green), vWF (magenta), and FH (cyan). In untreated samples, co-localization of TSP-1 and vWF was observed with limited involvement of C3 and FH. However, stimulation with S1 protein resulted in a significant increase in TSP-1/vWF fibers, accompanied by robust co-localized deposition of C3 and FH. Scale Bar = 50 µm.

Article Snippet: Human TSP-1 cDNA was obtained from Sinobiological (HG10508-UT) and subcloned into a pFastbac1 vector containing a gp67 signal peptide for secretion and an N-terminal 10xHis tag for purification.

Techniques: Immunostaining

Journal: bioRxiv

Article Title: Thrombospondin-1 inhibits alternative complement pathway activation in vasculitis synergistically to factor H

doi: 10.1101/2024.07.31.606000

Figure Lengend Snippet: TSP-1 is involved in the pathogenesis of ANCA vasculitis. TSP-1 is significantly increased in crescents of ANCA-vasculitis patients. Representative images of TSP-1 IHC staining of kidney biopsies and cancer nephrectomies. Pronounced TSP-1 staining can be seen in glomerular crescents (red dashed lines) of four ANCA-vasculitis patients (ANCA) while TSP-1 is absent in healthy controls or patients with focal segmental glomerulosclerosis (FSGS; black dashed lines indicate sclerosis). Scale Bar = 50 µm.

Article Snippet: Human TSP-1 cDNA was obtained from Sinobiological (HG10508-UT) and subcloned into a pFastbac1 vector containing a gp67 signal peptide for secretion and an N-terminal 10xHis tag for purification.

Techniques: Immunohistochemistry, Staining

Journal: bioRxiv

Article Title: Thrombospondin-1 inhibits alternative complement pathway activation in vasculitis synergistically to factor H

doi: 10.1101/2024.07.31.606000

Figure Lengend Snippet: TSP-1 regulates complement activity as well as neutrophil extracellular trap release in an ANCA-vasculitis model. (A) Blockade of TSP-1 in ANCA-PR3 treated whole blood causes pronounced release of DNA NETs. HUVECs coated µ-slides were perfused with whole blood treated with hirudin and with ANCA-PR3 antibodies isolated from an ANCA-vasculitis patient (patient ANCA1). Whole blood was stained with DAPI and NET release monitored for 4 hours via live cell imaging. Treatment of whole blood did not cause visible NET release. ANCA-PR3 antibodies in combination with TSP-1 blockade caused pronounced NET release on endothelial cells. In contrast, ANCA-PR3 antibodies in combination with a TSP-1 isotype control antibody did not have an effect. Scale Bar = 100 µm (B) Antibody mediated blockade of TSP-1 modulates plasma C5a levels as well as the release of histone-DNA complexes in a microfluidic experiment mimicking ANCA-vasculitis. ANCA-PR3 antibodies induce a significant increase of plasma TSP-1 levels which can be suppressed by addition of a TSP-1 antibody. The addition of ANCA-PR3 antibodies to whole blood leads to a substantial increase in plasma C5a levels compared to untreated whole blood (Neg. ctrl.). This increase is further amplified when combined with a TSP-1 antibody. Treatment of whole blood with ANCA-PR3 antibodies results in a significant rise in released histone-DNA complexes. This increase is further intensified when TSP-1 antibody is added. Bars represent means ± SD of 3 independent experiments. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0,001, One-way ANOVA using Turkey’s multiple comparison test. (C) Blockade of TSP-1 causes increased C3 deposition on HUVECs perfused with ANCA treated whole blood. Treatment of whole blood with ANCA-PR3 causes a notable enhancement of both C3 and TSP-1 deposition on HUVECs compared to untreated samples (Neg. ctrl.). Furthermore, the increase in C3 deposition is significantly augmented when combined with a TSP-1 antibody. Mean fluorescence analysis of C3 staining is presented on the right. Bars represent means ± SD of 3 independent experiments. *P ≤ 0.05, ***P ≤ 0.01, One-way ANOVA using Turkey’s multiple comparison test. Scale Bar = 50 µm.

Article Snippet: Human TSP-1 cDNA was obtained from Sinobiological (HG10508-UT) and subcloned into a pFastbac1 vector containing a gp67 signal peptide for secretion and an N-terminal 10xHis tag for purification.

Techniques: Activity Assay, Isolation, Staining, Live Cell Imaging, Control, Amplification, Comparison, Fluorescence

Journal: bioRxiv

Article Title: Thrombospondin-1 inhibits alternative complement pathway activation in vasculitis synergistically to factor H

doi: 10.1101/2024.07.31.606000

Figure Lengend Snippet: Summary of TSP-1 effects on the alternative pathway and physiological significance. (A) Complement inhibitory functions of TSP-1 in comparison to factor H are schematically illustrated: TSP-1 prevents cleavage of FB by FD in vitro, although binding to FB has not been confirmed by SPR and therefore not physiologically relevant (dashed line). TSP-1 binds to C3 and prevents the cleavage of C3 into C3a and C3b. Furthermore, TSP-1 binds to C5 and prevents its cleavage into C5a and C5b and the formation of the MAC. Possible physiological and pathophysiological complement regulatory functions of TSP-1 in simplified models: secondary complement activation occurs in SARS-CoV-2 hyperinflammation and thrombosis (B) due to COVID spike protein induced endothelial impairment and thrombotic events related to the formation of ultra large vWF multimers and in ANCA-vasculitis (C) due to neutrophil activation, netosis and subsequent vasculitis. In these local overwhelming conditions, inhibition by FH might not be sufficient to control complement activation. Therefore, additional complement inhibitory functions by locally released TSP-1 from endothelia and/or thrombocytes could be physiologically relevant regarding control of excessive complement activation especially on surfaces.

Article Snippet: Human TSP-1 cDNA was obtained from Sinobiological (HG10508-UT) and subcloned into a pFastbac1 vector containing a gp67 signal peptide for secretion and an N-terminal 10xHis tag for purification.

Techniques: Comparison, In Vitro, Binding Assay, Activation Assay, Inhibition, Control

Journal: Advanced Science

Article Title: Single‐Cell RNA Sequencing and Spatial Transcriptomics Reveal Pathogenesis of Meningeal Lymphatic Dysfunction after Experimental Subarachnoid Hemorrhage

doi: 10.1002/advs.202301428

Figure Lengend Snippet: Intercellular communication networks in and around mLVs. A) Spatial visualization of immune cell infiltration based on mLECs distribution. B) Outgoing and incoming signal patterns among all meningeal cells. C) THBS signaling pathway network among all cell types. D) Spatial visualization of THBS1‐CD47 L‐R pair distribution by stLearn. E) Volcano plots showing that THBS1 is one of the upregulated DEGs in the SAH group compared to the NPH group identified by mass spectrometry. F) Quantification of THBS1, THBS2, and THBS4 expression in CSF samples from SAH patients compared to those from NPH patients by ELISA assay, *** p < 0.001 versus NPH by paired two‐tailed Student's t ‐test. G) Representative confocal images of mLVs region of the negative control (NC) group and recombinant THBS1 protein treatment (rTHBS1) group. An enlarged view of TS and SSS is shown on the right in each group. Lyve1 (blue), Beads (green), and CD31 (red). Scale bar: 1000 µm. H) Flow cytometric analysis of mLECs percentage in NC group and rTHBS1 group, n = 4 per group, *** p < 0.001 versus NC by paired two‐tailed Student's t ‐test. I) Representative images of beads accumulation in dCLNs of the NC group and rTHBS1 group. Scale bar: 200 µm.

Article Snippet: The concentrations of human CSF THBS1 (EK0899, Boster Biotech, Wuhan), THBS2 (EK0642, Boster Biotech, Wuhan), THBS4 (CSB‐EL023490HU, Cusabio Biotech, Wuhan), and S100A6 (CSB‐EL020634RA, Cusabio Biotech, Wuhan) were determined using ELISA kits.

Techniques: Mass Spectrometry, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Negative Control, Recombinant

Journal: Advanced Science

Article Title: Single‐Cell RNA Sequencing and Spatial Transcriptomics Reveal Pathogenesis of Meningeal Lymphatic Dysfunction after Experimental Subarachnoid Hemorrhage

doi: 10.1002/advs.202301428

Figure Lengend Snippet: Cell trajectory analysis shows the evolution of mLECs. A) Labeling mLECs with real grouping information based on pseudotime trajectory map. B) Pseudotime trajectory map. C) Trajectory of representative genes in mLECs. D) Heatmap showing the top 30 pseudotime‐related genes. E) Representative two pseudo‐time‐related genes (S100 α 6 and Cldn5) based on q values. F,G) Increased expression of THBS1 and S100A6 are associated with poor prognosis, n = 48, * p < 0.05 by paired two‐tailed Student's t ‐test. H) Linear relationship between THBS1 and S100A6 expression, r = 0.3779, p = 0.0081 by Spearman's rank test. H) Representative confocal images of mLVs region of sham and SAH 24 h group. Enlarged view of selected region in the merged photo (yellow dotted box) are listed on the right in each group. Lyve1 (blue), S100 α 6 (green), and CD31 (red). Yellow arrows indicate where mLVs lie. Scale bar: 800 µm for the holistic view and 100 µm for the enlarged view.

Article Snippet: The concentrations of human CSF THBS1 (EK0899, Boster Biotech, Wuhan), THBS2 (EK0642, Boster Biotech, Wuhan), THBS4 (CSB‐EL023490HU, Cusabio Biotech, Wuhan), and S100A6 (CSB‐EL020634RA, Cusabio Biotech, Wuhan) were determined using ELISA kits.

Techniques: Labeling, Expressing, Two Tailed Test

Journal: Advanced Science

Article Title: Single‐Cell RNA Sequencing and Spatial Transcriptomics Reveal Pathogenesis of Meningeal Lymphatic Dysfunction after Experimental Subarachnoid Hemorrhage

doi: 10.1002/advs.202301428

Figure Lengend Snippet: Validation of THBS1 impacting mLVs function after SAH. SAH modeling and behavioral tests were performed 4 weeks after the AAV injection. A) Flow cytometric analysis of mLECs percentage in Sham, AAV‐control, and AAV‐THBS1 group at 72 h post‐SAH, n = 4 per group, ** p < 0.01, *** p < 0.001 by paired two‐tailed Student's t ‐test. B) Representative images and quantification of beads accumulation in dCLNs of Sham, AAV‐control, and AAV‐THBS1 group at 72 h post‐SAH, each data point represents an average of the 2 dCLNs from one individual mouse, n = 8 per group, ** p < 0.01, *** p < 0.001 by paired two‐tailed Student's t ‐test. Scale bar: 200 µm. C) Representative confocal images of mLVs region of Sham, AAV‐control, and AAV‐THBS1 group at 72 h post‐SAH. An enlarged view of TS and SSS is shown on the right in each group. Lyve1 (blue), Beads (green), and CD31 (red). Scale bar: 1000 µm. D) Modified Garcia test, E) time turn, F) time total, and G) wire hanging test at 72 h after SAH revealed AAV‐THBS1 delivery aggravated short‐term neurological function compared with Sham or AAV‐control group, n = 10–12 per group. * p < 0.05; ** p < 0.01, *** p < 0.001 by paired two‐tailed Student's t ‐test. H) Flow cytometric analysis of mLECs percentage in Sham, SAH‐WT, and THBS1‐KO group at 24 h post‐SAH, n = 4 per group, ** p < 0.01, *** p < 0.001 by paired two‐tailed Student's t ‐test. I) Representative images and quantification of beads accumulation in dCLNs in sham, SAH‐WT, and THBS‐KO group at 24 h post‐SAH, each data point represents an average of the 2 dCLNs from one individual mouse. n = 8 per group, ** p < 0.01 by paired two‐tailed Student's t ‐test. Scale bar: 200 µm. J) Representative confocal images of mLVs region of Sham, SAH‐WT, and THBS1‐KO group at 24 h post‐SAH. An enlarged view of TS and SSS is shown on the right in each group. Lyve1 (blue), Beads (green), and CD31 (red). Scale bar: 1000 µm. K) Modified Garcia test, L) time turn, M) time total, and N) wire hanging test at 24 h after SAH revealed THBS1 knockout improved short‐term neurological function compared with Sham or WT‐SAH group. n = 10–12 per group. * p < 0.05; ** p < 0.01, *** p < 0.001 by paired two‐tailed Student's t ‐test.

Article Snippet: The concentrations of human CSF THBS1 (EK0899, Boster Biotech, Wuhan), THBS2 (EK0642, Boster Biotech, Wuhan), THBS4 (CSB‐EL023490HU, Cusabio Biotech, Wuhan), and S100A6 (CSB‐EL020634RA, Cusabio Biotech, Wuhan) were determined using ELISA kits.

Techniques: Biomarker Discovery, Injection, Control, Two Tailed Test, Modification, Knock-Out

Journal: Advanced Science

Article Title: Single‐Cell RNA Sequencing and Spatial Transcriptomics Reveal Pathogenesis of Meningeal Lymphatic Dysfunction after Experimental Subarachnoid Hemorrhage

doi: 10.1002/advs.202301428

Figure Lengend Snippet: Disturbing THBS1‐CD47 interaction promoted mLVs restoration via inhibiting STAT3/BCL2‐mediated apoptosis in mLECs. A) Flow cytometric analysis of mLECs percentage in sham, SAH + igG, SAH + anti‐CD47, and SAH + anti‐THBS1 group at 24 h post‐SAH, n = 4 per group, *** p < 0.001 by paired two‐tailed Student's t ‐test. B) Representative images and quantification of beads accumulation in dCLNs of Sham, SAH + igG, SAH + anti‐CD47, and SAH + anti‐THBS1 group at 24 h post‐SAH, each data point represents an average of the 2 dCLNs from one individual mouse, n = 8 per group, *** p < 0.001 by paired two‐tailed Student's t ‐test. Scale bar: 200 µm. C) Representative confocal images of mLVs region of Sham, SAH + igG, SAH + anti‐CD47, and SAH + anti‐THBS1 group at 24 h post‐SAH. An enlarged view of TS and SSS is shown on the right in each group. Lyve1 (blue), Beads (green), and CD31 (red). Scale bar: 1000 µm. D) Modified Garcia test, E) time turn, F) time total, and G) wire hanging test at 24 h after SAH revealed anti‐CD47 and anti‐THBS1 therapy improved short‐term neurological function compared with Sham or SAH + igG group. n = 10–12 per group. * p < 0.05; ** p < 0.01, *** p < 0.001 by paired two‐tailed Student's t ‐test. H) GSEA showed apoptosis pathway was activated in mLECs after SAH. I) Representative immunoblot images showing effects of rTHBS1 (100 ng mL −1 ) treatment on pSTAT3 and Bcl‐2 inhibition in primary mLECs. J–M) Representative immunoblot images of STAT3, pSTAT3, Bax, and Bcl‐2 in different groups.

Article Snippet: The concentrations of human CSF THBS1 (EK0899, Boster Biotech, Wuhan), THBS2 (EK0642, Boster Biotech, Wuhan), THBS4 (CSB‐EL023490HU, Cusabio Biotech, Wuhan), and S100A6 (CSB‐EL020634RA, Cusabio Biotech, Wuhan) were determined using ELISA kits.

Techniques: Two Tailed Test, Modification, Western Blot, Inhibition

Journal: The Journal of Clinical Investigation

Article Title: Thrombospondin 1 missense alleles induce extracellular matrix protein aggregation and TM dysfunction in congenital glaucoma

doi: 10.1172/JCI156967

Figure Lengend Snippet: ( A ) Pedigrees of 3 families with THBS1 mutations. Specific mutations in THBS1 are listed below the family number, with carrier family members annotated as +/M. Affected individuals are indicated by solid black symbols. Note: White symbols do not exclude undiagnosed late-onset disease. ( B ) Schematic representation of THBS1 protein domains and location of R1034 in the C-terminal domain. R1034 is the first amino acid of an 8–amino acid sequence involved with CD47 binding. VWFC, von Willebrand factor type C domain. ( C ) Sequence alignment of THBS1 R1034 showing strong evolutionary conservation.

Article Snippet: The following plasmids containing cDNA coding for THBS1 with the N-terminal FLAG tag were purchased from Sino Biological for mutagenesis: human THBS1 cDNA ORF clone FLAG tag (catalog HG10508-NF, Sino Biological) and mouse Thbs1 cDNA ORF Clone FLAG tag (catalog MG50655-NF, Sino Biological).

Techniques: Sequencing, Binding Assay

Journal: The Journal of Clinical Investigation

Article Title: Thrombospondin 1 missense alleles induce extracellular matrix protein aggregation and TM dysfunction in congenital glaucoma

doi: 10.1172/JCI156967

Figure Lengend Snippet: ( A ) CRISPR/Cas9 gene editing was used to generate the R1034C mutation in murine Thbs1 . The R1034 sequence codon is conserved between humans and mice. ( B ) Strategy for single-stranded oligo DNA nucleotide–mediated (ssODN-mediated) knockin with CRISPR/Cas9. Successful mutagenesis generated a PuvII digestion site (black arrowhead) ( C ). Mutants were confirmed by the presence of 242 and 211 bp bands following PCR and PuvII digestion. ( D ) Sanger sequencing confirmed the F1 homozygous versus littermate WT from heterozygous founders in C . RNA purified from F1 homozygous mutants also confirmed the point mutation .

Article Snippet: The following plasmids containing cDNA coding for THBS1 with the N-terminal FLAG tag were purchased from Sino Biological for mutagenesis: human THBS1 cDNA ORF clone FLAG tag (catalog HG10508-NF, Sino Biological) and mouse Thbs1 cDNA ORF Clone FLAG tag (catalog MG50655-NF, Sino Biological).

Techniques: CRISPR, Mutagenesis, Sequencing, Knock-In, Generated, Purification

Journal: The Journal of Clinical Investigation

Article Title: Thrombospondin 1 missense alleles induce extracellular matrix protein aggregation and TM dysfunction in congenital glaucoma

doi: 10.1172/JCI156967

Figure Lengend Snippet: ( A ) IOP measurements in Thbs1 R1034C homozygous and heterozygous mutant mice. Compared with age-matched WT controls, both homozygous ( P = 0.0016) and heterozygous ( P = 0.007) mutant mice showed elevated IOP. Heterozygous, n = 14; homozygous, n = 18; controls, n = 24. Data represent the mean ± SEM. P values were determined by repeated-measures 1-way ANOVA followed by Bonferroni’s correction. ( B ) In vivo measurement of aqueous humor outflow facility (reciprocal of outflow resistance) in 4-month-old mice. Thbs1 R1034C -mutant mice showed a significant reduction of outflow facility compared with WT controls. Heterozygous, n = 9; heterozygous controls, n = 9; homozygous, n = 9; homozygous controls, n = 9. *** P ≤ 0.01, by 2-tailed Student’s t test followed by Bonferroni’s correction. The box boundaries extend from the 25th to the 75th percentiles, the line within the boxes are the mean values, and the whiskers indicate the minimum and maximum values. ( C ) RGC density was determined by BRN3A staining on retinal whole mounts. RGC counts from 6 central and 12 peripheral AOI (AOI = 0.04 mm 2 ) were averaged for each eye. Scale bar: 1.0 mm. ( D ) Representative AOI images from C . Scale bar: 50 μm; insets, ×0.32. ( E ) Compared with WT mice, there was a significant reduction of RGC density in both the central and peripheral retinas of homozygous (15.9% and 22.2%, respectively, n = 8) and heterozygous (5.3% and 10.1%, respectively, n = 8) animals. Data represent the mean ± SEM. ** P = 0.007 and **** P ≤ 0.0001, by 2-way ANOVA.

Article Snippet: The following plasmids containing cDNA coding for THBS1 with the N-terminal FLAG tag were purchased from Sino Biological for mutagenesis: human THBS1 cDNA ORF clone FLAG tag (catalog HG10508-NF, Sino Biological) and mouse Thbs1 cDNA ORF Clone FLAG tag (catalog MG50655-NF, Sino Biological).

Techniques: Mutagenesis, In Vivo, Staining

Journal: The Journal of Clinical Investigation

Article Title: Thrombospondin 1 missense alleles induce extracellular matrix protein aggregation and TM dysfunction in congenital glaucoma

doi: 10.1172/JCI156967

Figure Lengend Snippet: ( A ) THBS1-stained cryosections of the iridocorneal angle region from 4-month-old Thbs1 R1034C homozygous mice and age-matched controls. TM was localized adjacent to the CD31-stained SC. Thbs1 R1034C -mutant mice showed increased THBS1 protein expression in TM compared with WT controls. Nuclei were stained with DAPI. Scale bar: 100 μm; insets, ×2.4. ( B ) IF staining for THBS1 in corneal whole mounts showing an en face view of protein localization. SC is indicated by dashed lines, as revealed by CD31 staining. Scale bar: 200 μm; insets, ×0.23 (upper left in B and C ). ( C ) Progressive THBS1 protein accumulation in TM was evident in homozygous mice, and a similar pattern of accumulated THBS1 protein was evident in Thbs R1034C heterozygous mice. Scale bar: 200 μm; insets, ×3.6 (lower right in B and C ). ( D ) Quantification of THBS1 in TM based on fluorescence intensity (100 μm 2 fields were captured for intensity measurements; n = 9 for each time point). AU, arbitrary fluorescence units. Data represent the mean ± SEM. ( E ) Thbs1 R1034C homozygous mice did not show increased Thbs1 mRNA expression (as determined by unpaired 2-tailed Student’s t test). Relative mRNA levels were normalized to Gapdh . n = 3. Data represent the mean ± SEM. ( F ) Western blot of protein extracts from the corneal limbal ring containing the iridocorneal angle region. Higher levels of THBS1 protein were detected in homozygous Thbs1 R1034C mice, however, there was no evidence of ER stress in the mutant mice at age 16 months of age compared with age-matched controls based on the expression of GRP94, IRE1A, and BIP.

Article Snippet: The following plasmids containing cDNA coding for THBS1 with the N-terminal FLAG tag were purchased from Sino Biological for mutagenesis: human THBS1 cDNA ORF clone FLAG tag (catalog HG10508-NF, Sino Biological) and mouse Thbs1 cDNA ORF Clone FLAG tag (catalog MG50655-NF, Sino Biological).

Techniques: Staining, Mutagenesis, Expressing, Fluorescence, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Thrombospondin 1 missense alleles induce extracellular matrix protein aggregation and TM dysfunction in congenital glaucoma

doi: 10.1172/JCI156967

Figure Lengend Snippet: ( A ) Abnormal ECM deposition was evident in 2-month-old Thbs1 R1034C -mutant mice (homozygous and heterozygous) compared with WT controls, primarily in the JCT adjacent to SC. In mutant mice, a dense fibrogranular material was present in the ECM but was not evident in WT mice, which exhibited organized collagen fibers. Scale bar: 1 μm; enlarged insets, ×5. ( B ) Gold particle labeling was combined with electron microscopy to localize THBS1 protein in TM at the ultrastructural level. Gold particles labeled aggregated THBS1 R1034C deposits (small black dots). Scale bars: 0.125 μm. ( C ) Loss of cellularity was evident in the JCT cells of mutant mice, presumably due to the aggregation of THBS1 R1034C . Scale bar: 1 μm. GV, giant vacuole.

Article Snippet: The following plasmids containing cDNA coding for THBS1 with the N-terminal FLAG tag were purchased from Sino Biological for mutagenesis: human THBS1 cDNA ORF clone FLAG tag (catalog HG10508-NF, Sino Biological) and mouse Thbs1 cDNA ORF Clone FLAG tag (catalog MG50655-NF, Sino Biological).

Techniques: Mutagenesis, Labeling, Electron Microscopy

Journal: The Journal of Clinical Investigation

Article Title: Thrombospondin 1 missense alleles induce extracellular matrix protein aggregation and TM dysfunction in congenital glaucoma

doi: 10.1172/JCI156967

Figure Lengend Snippet: ( A ) The C-terminal of human THBS1 consists of type 3 repeats 4–7 and a lectin-like domain. Arg1034 (orange) in the lectin-like domain interacts with repeat 5 (blue) by intramolecular forces, such as electrostatic and hydrogen bonds. ( B ) Cysteine, however, cannot sustain these interactions due to a shorter and uncharged side chain. ( C ) As a result, the R1034C substitution significantly destabilizes the C-terminal structure (mean ddGB = 2.17 kcal/mol). Of note, the destabilizing effect is dramatically eliminated upon the exclusion of repeat 5 from the C-terminal, indicating the important role of repeat 5 and Arg1034 in protein structure stabilization. ddGB was derived from 5 independent protein stability analyses on FoldX. Data represent the mean ± SEM.

Article Snippet: The following plasmids containing cDNA coding for THBS1 with the N-terminal FLAG tag were purchased from Sino Biological for mutagenesis: human THBS1 cDNA ORF clone FLAG tag (catalog HG10508-NF, Sino Biological) and mouse Thbs1 cDNA ORF Clone FLAG tag (catalog MG50655-NF, Sino Biological).

Techniques: Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: Thrombospondin 1 missense alleles induce extracellular matrix protein aggregation and TM dysfunction in congenital glaucoma

doi: 10.1172/JCI156967

Figure Lengend Snippet: ( A ) The propensity for THBS1 R1034C aggregation in the ECM was further assessed through ectopic expression of FLAG-tagged THBS1 in COS-7 cells. The absence of DAPI (nuclear acid) and F-actin (cytoskeleton) staining indicated the successful removal of cellular components. THBS1 extracellular deposition was evaluated by anti-FLAG IF staining. Consistent with the in vivo findings, there was greater accumulation of mutant THBS1 C1034 in ECM than of THBS1 R1034 (WT). Scale bar: 10 μm; enlarged insets, ×4. ( B ) Plasmids with Arg1034 mutated to Lys, Gln, Ser, Ala, and Gly were transfected into COS-7 cells. ECM deposition was lowest in Lys1034 and greatest in Gly1034, with Gln, Ser, and Ala showing intermediate effects. Scale bar: 10 μm. ( C ) THBS1 ECM deposition strongly correlated with the change in protein stability ( R 2 = 0.86, P < 0.0001, by linear regression), suggesting that Arg1034 had an essential role in reducing abnormal THBS1 ECM deposition. The change in protein stability (ddGB) was derived from 5 analyses in FoldX. Protein deposition was quantified on the basis of fluorescence intensity of anti-FLAG immunostaining from 3 independent experiments. The mean values of ddGB and fluorescence intensity were used for regression analysis. Error bar indicates the SEM.

Article Snippet: The following plasmids containing cDNA coding for THBS1 with the N-terminal FLAG tag were purchased from Sino Biological for mutagenesis: human THBS1 cDNA ORF clone FLAG tag (catalog HG10508-NF, Sino Biological) and mouse Thbs1 cDNA ORF Clone FLAG tag (catalog MG50655-NF, Sino Biological).

Techniques: Expressing, Staining, In Vivo, Mutagenesis, Transfection, Derivative Assay, Fluorescence, Immunostaining

Journal: The Journal of Clinical Investigation

Article Title: Thrombospondin 1 missense alleles induce extracellular matrix protein aggregation and TM dysfunction in congenital glaucoma

doi: 10.1172/JCI156967

Figure Lengend Snippet: Costaining for THBS1 (green) and ( A ) RN, ( B ) COL1, ( C ) COL4, or ( D ) LAMA1 (magenta) in cryosections of the iridocorneal angle region. THBS1 R1034C colocalized (yellow arrowheads) with FN and to a lesser extent with COL1, COL4, and LAMA1. Nuclei were stained with DAPI. Scale bars: 20 μm. Insets: ×1.6.

Article Snippet: The following plasmids containing cDNA coding for THBS1 with the N-terminal FLAG tag were purchased from Sino Biological for mutagenesis: human THBS1 cDNA ORF clone FLAG tag (catalog HG10508-NF, Sino Biological) and mouse Thbs1 cDNA ORF Clone FLAG tag (catalog MG50655-NF, Sino Biological).

Techniques: Staining

Journal: The Journal of Clinical Investigation

Article Title: Thrombospondin 1 missense alleles induce extracellular matrix protein aggregation and TM dysfunction in congenital glaucoma

doi: 10.1172/JCI156967

Figure Lengend Snippet: Costaining for THBS1 (green) and ( A ) FN, ( B ) COL1, ( C ) COL4, and ( D ) LAMA1 (magenta) in the ECM in COS-7 cells. Consistent with the findings in TM, there was strong accumulation of FN, which colocalized with THBS1 C1034 . Additionally, there was moderate accumulation of COL1. Scale bars: 2 μm. Insets: ×1.6.

Article Snippet: The following plasmids containing cDNA coding for THBS1 with the N-terminal FLAG tag were purchased from Sino Biological for mutagenesis: human THBS1 cDNA ORF clone FLAG tag (catalog HG10508-NF, Sino Biological) and mouse Thbs1 cDNA ORF Clone FLAG tag (catalog MG50655-NF, Sino Biological).

Techniques: