Fig 2: SPARCL1 gene ko promotes acute and chronic DSS-induced colonic inflammation. A, Schematic overview of DSS-induced (acute and chronic) colitis with timepoints of DSS application, endoscopy, and intravital microscopy. The severity of colitis in Sc1 ko (n = 10) and WT (n = 10) mice was evaluated by (B) the endoscopic score in the acute colitis model. C, Survival analysis (left panel) and endoscopic score (right panel) of the Sc1 ko and WT (n=10 each) mice during the chronic colitis model (scores separated for each cycle). D, Histological analysis using H&E staining of the Sc1 ko and WT mice in the acute and chronic colitis model (left panel, scale bar 250 µm). Inserts show fibrosis (left insert) and tertiary lymphoid structures in the chronic model (right insert, scale bar 100 µm). Quantification of the amount of acute and chronic inflammation as detected by H&E staining (right panel). **P < 0.01, ***P < 0.001 by 2-tailed unpaired Student t test. H&E indicates hematoxylin-eosin.

Fig 3: SPARCL1 is expressed in blood vessels of metatarsals and inhibits angiogenesis ex vivo. A, Tissues covering metatarsals of WT embryos at E18.5 (n = 4) were stained for SPARCL1 (green), CD31 (red, upper), and αSMA (red, lower). Examples of co-localization of SPARCL1 and CD31 indicated by arrows and examples of co-localization of SPARCL1 and αSMA indicated by arrowheads. Draq5 [1, 5-bis{[2-(di-methylamino)ethyl]amino}-4, 8-dihydroxyanthracene-9, 10-dione] was used as counterstain. Scale bar: 50 μm. B, WT metatarsals of embryos at E18.5 were either untreated (n = 17), treated with recombinant murine SPARCL1 (mSPARCL1, 1.5 μg/mL; n = 8), or treated with vascular endothelial growth factor (100 ng/mL; n = 11) alone or in combination with mSPARCL1 (n = 10). Vessel endothelial cells are visualized by CD31 staining. One representative picture of 3 experiments is depicted. Scale bar: 500 μm. Quantification of relative numbers of branches and junctions includes pooled results of all experiments. Error bars indicate SD. *P < 0.05 and ****P < 0.0001 by 2-tailed, unpaired Student t test.

Fig 4: The AD of soluble SPARCL1 is required for its anti-angiogenic activity. A, Sc1 WT protein and mutants lacking specific domains of SPARCL1 (?AD, ?FLD, ?ECBD) with a signal peptide for secretion and a Flag-Glycine(9)-His-Tag for purification were expressed in HeLa cells. Secreted proteins were harvested from the cell culture supernatants and subjected to His-Tag chromatography. B, Coomassie stain of FPLC purified proteins in comparison to a commercially available purified WT SPARCL1 (200 ng each) and body surface area as reference. C, WT metatarsals of embryos at E18.5 were either left untreated (n = 10) or were treated with VEGF (100 ng/mL) (n = 11) alone or in combination with recombinant hSPARCL1 (1.5 µg/mL; n = 11) on days 3, 5, 7, and 9. SPARCL1 neutralization was performed using the hSPARCL1 antibody (a-SPARCL1; n = 10) in a 5-fold molar concentration and the respective isotype antibody (a-isotype; n = 9) as a control. Vessel endothelial cells were stained with CD31. One representative picture of 2 experiments is shown. Quantification of relative numbers of branches and junctions includes pooled results of all experiments. D, Similar to panel C, WT metatarsals were either untreated (n = 6) or treated with VEGF (100 ng/mL; n = 13) either alone or in combination with hSPARCL1 (1.5 µg/mL = 11.1 nM; n = 7), ?AD (11.1 nM; n = 6), ?FLD (11.1 nM; n = 5), or ?ECBD (11.1 nM; n = 7). Vessel endothelial cells are visualized by CD31 staining. One representative picture is shown. Relative numbers of branches and junctions are quantified. C and D, Scale bars: 500 µm. Error bars indicate SD. *P < 0.05, **P < 0.01, ***P < 0.001 by 2-tailed unpaired Student t test. hSPARCL1 indicates human SPARCL1; VEGF, vascular endothelial growth factor.

Fig 5: SPARCL1 is required for vessel morphogenesis and integrity. A, Sc1 ko (n = 6) and WT (n = 11) metatarsals of embryos at E18.5 were treated with vascular endothelial growth factor (100 ng/mL). Vessel endothelial cells are visualized by CD31 staining, and 1 representative picture is shown. Scale bar: 500 μm. Mean of vessel signal per area and number of intervessel gaps per area were quantified. In total, 20 increments surrounding the bones were defined with an area of 600,000 pixels per increment, starting with increment 1 as the first next to the bone (compare scheme, top right). Error bars indicate SD. *P < 0.05, **P < 0.01 by 2-tailed unpaired Student t test. B, Sc1 ko (n = 7) and WT (n = 7) mice were subjected to intravital microscopy of the colon. FITC-dextran was used to visualize vessels and the permeability of the crypts through FITC-dextran accumulation inside the crypts (green; examples indicated by arrows). Lectin was used to counterstain vessels only (red). Scale bar: 50 μm. Relative numbers of healthy crypts compared to leaky ones were quantified. In total, 1167 Sc1 ko and 723 WT crypts were counted and evaluated. ***P < 0.001 by χ 2 test.