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