Journal: Experimental and Therapeutic Medicine

Article Title: LSKL peptide alleviates subarachnoid fibrosis and hydrocephalus by inhibiting TSP1-mediated TGF-β1 signaling activity following subarachnoid hemorrhage in rats

doi: 10.3892/etm.2016.3640

Figure Lengend Snippet: LSKL peptide inhibited TSP1-mediated TGF-β1 signaling activity following SAH. Quantitative analyses of (A) TSP1 and (B) total TGF-β1 and active TGF-β1 in the CSF on days 3–5 after SAH. (C) Ratio of active TGF-β1 to total TGF-β1 in the CSF on days 3–5 after SAH. (D) Representative western blot bands of p-Smad2/3 and (E) quantitative analyses of p-Smad2/3 expression on day 5 after SAH. Relative densities of each protein have been normalized against the sham group. Data are expressed as the mean ± standard error of the mean (n=10). *P<0.05 vs. the sham group; # P<0.05 vs. the SAH+PBS group. SAH, subarachnoid hemorrhage; CSF, cerebrospinal fluid; TSP1, thrombospondin-1; LSKL, leucine-serine-lysine-leucine; PBS, phosphate buffer solution.

Article Snippet: These CSF samples were mixed and divided into three equal shares for the detection of TSP1, activated TGF-β1 and total TGF-β1 using the respective ELISA kit (Wuhan Boster Biological Technology, Ltd., Wuhan, China).

Techniques: Activity Assay, Western Blot, Expressing

Journal: Nature Communications

Article Title: Fasting mimicking diet in mice delays cancer growth and reduces immunotherapy-associated cardiovascular and systemic side effects

doi: 10.1038/s41467-023-41066-3

Figure Lengend Snippet:

Article Snippet: Anti-mouse Granzyme B, FITC (REA226) , Miltenyi Biotec , 130-118-341.

Techniques: In Vivo

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