Journal: Scientific Reports

Article Title: Glycosaminoglycans influence enzyme activity of MMP2 and MMP2/TIMP3 complex formation - Insights at cellular and molecular level

doi: 10.1038/s41598-019-41355-2

Figure Lengend Snippet: Influence of CS, HA, SH, and HE on MMP2 and TIMP3 formation and MMP2 activity in hBMSC. 7,000 hBMSC/cm 2 were plated in basic medium and treated with CS, HA, SH, and HE (200 µg/mL each). At day 22 after plating cells and conditioned medium were analyzed. ( A ) mmp2 expression was assessed by qPCR, normalized to the expression of the house-keeping genes (hkg) gapdh , β-actin , and rps26 , and related to untreated control (Ctrl, set to 1) using the comparative quantitation method. Conditioned medium of hBMSC was analyzed for MMP2 protein content using commercially MMP2 ELISA Kit ( B ) and for MMP2 enzyme activity with fluorogenic peptide (MCA-Pro-Leu-Ala-Nva-Dpa-Ala-Arg-NH 2 ) as a substrate ( C ). ( D ) The relative MMP2 activity per MMP2 protein amount was calculated from fluorescence signals (MMP2 activity) and ELISA data (MMP2 protein). ( E ) timp3 expression was assessed by qPCR as described. ( F ) TIMP3 protein amount released into the conditioned medium was determined by ELISA. TIMP3 protein in the pericellular environment of hBMSC was analyzed by Western blotting with chemiluminescence detection. ( G ) shows a representative Western blot (cropped), according full-length blots presented in Supplemental Fig. . The chemiluminescence signals of four independent Western blots were quantified densitometrically and normalized to GAPDH and β-tubulin (Tub) as internal loading controls ( H ). ( I ) shows the total TIMP3 content related to Ctrl and the distribution of TIMP3 calculated from Western blot data (pericellular, ECM-associated TIMP3) and ELISA data (released TIMP3). The ratio of released MMP2 protein to released TIMP3 is shown in ( J ). The results are presented as mean ± SEM. Significant differences of treatment vs. Ctrl were analyzed by one-way ANOVA/Bonferroni’s post-test ( A – F , H ,J), and of SH vs. HE two-way ANOVA/Bonferroni’s post-test ( I ) and are indicated with *(p < 0.05), **(p < 0.01) and ***(p < 0.001), n = 4.

Article Snippet: MMP2 and TIMP3 protein concentration in conditioned medium of hBMSC was determined using DuoSet ELISA development kits (DY902 for MMP2, and DY973 for TIMP3, BioTechne, Wiesbaden, Germany) according to manufacturer’s instructions.

Techniques: Activity Assay, Expressing, Quantitation Assay, Enzyme-linked Immunosorbent Assay, Fluorescence, Western Blot

Journal: Scientific Reports

Article Title: Glycosaminoglycans influence enzyme activity of MMP2 and MMP2/TIMP3 complex formation - Insights at cellular and molecular level

doi: 10.1038/s41598-019-41355-2

Figure Lengend Snippet: Immunofluorescence staining and colocalization analysis of MMP2 and TIMP3, and of SH with MMP2 and TIMP3. 7,000 hBMSC/cm 2 were plated in basic medium and treated with CS, HA, SH, and HE (200 µg/mL each) ( A ) or with ATTO655-SH (violet) ( B ). At day 8 after plating cells were fixed with paraformaldehyde and stained for MMP2 (green), TIMP3 (red), and nuclei (blue); scale bars 50 µm ( A , B ). ( B ) Images in line from left to right show merged MMP2/TIMP3 (yellow), SH (violet), merged SH/MMP2 (white), merged SH/TIMP3 (purple), and merged SH/MMP2/TIMP3 (pale pink). The extent of colocalization of MMP2 with TIMP3 ( C ) (according to images in A ) and of SH with either MMP2 ( D ) or TIMP3 ( E ) (according to images in B ) was calculated as described. Pearson correlation coefficient (summarized signal) values >0.5 (dotted line) indicate a high probability that pixels of both channels are overlay. Manders’ coefficients M1 and M2 (M1 indicates the overlap of SH signal (violet channel) with MMP2 signal (green channel) ( D , E ). Values are given as mean ± SEM; significant differences of treatment vs. Ctrl were analyzed by one-way ANOVA/Bonferroni’s post-test and are indicated with ***(p < 0.001), n = 8.

Article Snippet: MMP2 and TIMP3 protein concentration in conditioned medium of hBMSC was determined using DuoSet ELISA development kits (DY902 for MMP2, and DY973 for TIMP3, BioTechne, Wiesbaden, Germany) according to manufacturer’s instructions.

Techniques: Immunofluorescence, Staining

Journal: Scientific Reports

Article Title: Glycosaminoglycans influence enzyme activity of MMP2 and MMP2/TIMP3 complex formation - Insights at cellular and molecular level

doi: 10.1038/s41598-019-41355-2

Figure Lengend Snippet: Influence of CS, HA, SH, and HE on MMP2 enzyme activity. MMP2 enzyme activity was determined with rhMMP2 (100 ng/mL) and 50 µM fluorogenic peptide (MCA-Pro-Leu-Ala-Nva-Dpa-Ala-Arg-NH 2 ) as a substrate. Kinetic measurement was performed for 5 h without GAG (black curve) and in the presence of CS ( A , green), HA ( B , brown), SH ( C , pink), and HE ( D , teal) (200 µg/mL each; 20–1,000 µg/mL shown in Supplemental Fig. ); blanks are given in light grey. ( E ) shows the endpoint values of MMP2 activity after 5 h; the horizontal line indicates the blank value. The results are presented as mean ± SEM. Significant differences of treatment vs. Ctrl were analyzed by one-way ANOVA/Bonferroni’s post-test and are indicated with ***(p < 0.001), n = 4.

Article Snippet: MMP2 and TIMP3 protein concentration in conditioned medium of hBMSC was determined using DuoSet ELISA development kits (DY902 for MMP2, and DY973 for TIMP3, BioTechne, Wiesbaden, Germany) according to manufacturer’s instructions.

Techniques: Activity Assay

Journal: Scientific Reports

Article Title: Glycosaminoglycans influence enzyme activity of MMP2 and MMP2/TIMP3 complex formation - Insights at cellular and molecular level

doi: 10.1038/s41598-019-41355-2

Figure Lengend Snippet: Influence of CS, HA, SH, and HE on TIMP3-induced MMP2 inhibition. MMP2 enzyme activity was determined with rhMMP2 (100 ng/mL) and 50 µM fluorogenic peptide (MCA-Pro-Leu-Ala-Nva-Dpa-Ala-Arg-NH 2 ) as a substrate. Kinetic measurement was performed for 5 h without TIMP3 (black curve) and in the presence of 40 ng rhTIMP3/mL ( A , blue curve); blanks are given in light grey. TIMP3-induced inhibition of MMP2 activity after 5 h (endpoint of kinetic measurement) is given in percent of MMP2 activity ( B ). In the presence of GAG (200 µg/mL) the activity assay was performed in different sequence: either MMP2 and TIMP3 or TIMP3 and GAG or MMP2 and GAG (given in parenthesis) were incubated for 30 min before adding the third component. ( C – E ) show how the sequence of incubating the assay components influenced the inhibitory effect of TIMP3 on MMP2 activity. The results are presented as mean ± SEM. Significant differences of treatment vs. control were analyzed by one-way ANOVA/Bonferroni’s post-test and are indicated with *(p < 0.05), **(p < 0.01), and ***(p < 0.001), n = 4. ( F – H ) Schematic representation of molecular recognition of GAG to MMP2/TIMP3 complex, TIMP3 and MMP2, respectively, and corresponding docking results using Autodock3 and DBSCAN clustering. MMP2 catalytic domain (PDB ID 1QIB (2.8 Å)) and TIMP3 model are shown in pale and blue cartoon, respectively. TIMP3 model in ( H ) and MMP2 in ( G ) are shown in blue and pale transparency, respectively, just for illustrative purposes, although not taken into account for docking calculations. Calcium and zinc ions are shown in green and grey spheres, respectively. Different clusters of GAG are shown in sticks with color gradient: SH ( G , H , pink) and HA ( F , brown). The cartoons above illustrate in a schematic manner the recognition of GAG to MMP2/TIMP3 complex, TIMP3 and MMP2 predicted by molecular modeling.

Article Snippet: MMP2 and TIMP3 protein concentration in conditioned medium of hBMSC was determined using DuoSet ELISA development kits (DY902 for MMP2, and DY973 for TIMP3, BioTechne, Wiesbaden, Germany) according to manufacturer’s instructions.

Techniques: Inhibition, Activity Assay, Sequencing, Incubation

Journal: Scientific Reports

Article Title: Glycosaminoglycans influence enzyme activity of MMP2 and MMP2/TIMP3 complex formation - Insights at cellular and molecular level

doi: 10.1038/s41598-019-41355-2

Figure Lengend Snippet: MM-GBSA binding free energies of MMP2/TIMP3 complex in the absence and presence of HA.

Article Snippet: MMP2 and TIMP3 protein concentration in conditioned medium of hBMSC was determined using DuoSet ELISA development kits (DY902 for MMP2, and DY973 for TIMP3, BioTechne, Wiesbaden, Germany) according to manufacturer’s instructions.

Techniques: Binding Assay

Journal: Scientific Reports

Article Title: Glycosaminoglycans influence enzyme activity of MMP2 and MMP2/TIMP3 complex formation - Insights at cellular and molecular level

doi: 10.1038/s41598-019-41355-2

Figure Lengend Snippet: Molecular modeling of GAG in complex with proMMP2. Docking results using Autodock3 and DBSCAN clustering are shown. proMMP2 (PDB ID 1GXD (3.1 Å)) is shown in cartoon and colored by domains: propeptide (light pink), catalytic domain (pale), FNII (green) and PEX domain (grey). Different clusters of GAG are shown in sticks with gradient colors: ( A ) C4S (green), ( B ) C6S (green), ( C ) HA (brown), ( D ) SH (pink) and ( E ) HE (teal). Calcium and zinc ions are shown in green and grey spheres, respectively. The TIMP3 model, although not taken into account for docking calculations, is shown in blue transparency just for illustrative purposes. Discontinuous black circles and rectangles highlight the PEX domain recognition by TIMP3 C-terminal tail and FNII, respectively.

Article Snippet: MMP2 and TIMP3 protein concentration in conditioned medium of hBMSC was determined using DuoSet ELISA development kits (DY902 for MMP2, and DY973 for TIMP3, BioTechne, Wiesbaden, Germany) according to manufacturer’s instructions.

Techniques:

Journal: Scientific Reports

Article Title: Glycosaminoglycans influence enzyme activity of MMP2 and MMP2/TIMP3 complex formation - Insights at cellular and molecular level

doi: 10.1038/s41598-019-41355-2

Figure Lengend Snippet: Molecular modeling of GAG in complex with proMMP2(PEX)/TIMP3. Docking results using Autodock3 and DBSCAN clustering of GAG (sticks) to PEX domain of proMMP2 (grey cartoon) (PDB ID 1GXD (3.1 Å)) in complex with TIMP3 model (blue cartoon). Different clusters of GAG are highlighted in gradient colors: ( A ) C4S (green) ( B ) C6S (green), ( C ) HA (brown), ( D ) SH (pink) and ( E ) HE (teal). The cartoon illustrates the molecular bridging of PEX and TIMP3 by SH (discontinuous circle) ( D ).

Article Snippet: MMP2 and TIMP3 protein concentration in conditioned medium of hBMSC was determined using DuoSet ELISA development kits (DY902 for MMP2, and DY973 for TIMP3, BioTechne, Wiesbaden, Germany) according to manufacturer’s instructions.

Techniques:

Journal: ACS Omega

Article Title: Mechanisms and Molecular Targets of BuShenHuoXue Formula for Osteoarthritis

doi: 10.1021/acsomega.1c07270

Figure Lengend Snippet: Relative mRNA expression rates of genes in chondrocytes by PCR. (A) SELE. (B) MMP2. (C) COL1. * P < 0.05, ** P < 0.01, *** P < 0.001 versus the IL-1β group.

Article Snippet: The following antibodies and corresponding dilution ratios were used: anti-SELE (bs-1273R, Rabbit, Bioss, 1:500, secondary antibody: 1:5000), anti-MMP2 (bs-0412R, Rabbit, Bioss, 1:500, secondary antibody: 1:5000), anti-CoL1 (bs-10423R, Rabbit, Bioss, 1:500, secondary antibody: 1:5000), and anti-GAPDH (60004-1-lg, Mouse, Proteintech, 120,000, secondary antibody, 1:5000).

Techniques: Expressing

Journal: ACS Omega

Article Title: Mechanisms and Molecular Targets of BuShenHuoXue Formula for Osteoarthritis

doi: 10.1021/acsomega.1c07270

Figure Lengend Snippet: Western blot in five groups of chondrocytes. (A) SELE. (B) MMP2. (C) COL1. (D) Electrophoretic images. ** P < 0.01, *** P < 0.001 versus IL-1β group.

Article Snippet: The following antibodies and corresponding dilution ratios were used: anti-SELE (bs-1273R, Rabbit, Bioss, 1:500, secondary antibody: 1:5000), anti-MMP2 (bs-0412R, Rabbit, Bioss, 1:500, secondary antibody: 1:5000), anti-CoL1 (bs-10423R, Rabbit, Bioss, 1:500, secondary antibody: 1:5000), and anti-GAPDH (60004-1-lg, Mouse, Proteintech, 120,000, secondary antibody, 1:5000).

Techniques: Western Blot

Journal: ACS Omega

Article Title: Mechanisms and Molecular Targets of BuShenHuoXue Formula for Osteoarthritis

doi: 10.1021/acsomega.1c07270

Figure Lengend Snippet: KEGG Enrichment Analysis

Article Snippet: The following antibodies and corresponding dilution ratios were used: anti-SELE (bs-1273R, Rabbit, Bioss, 1:500, secondary antibody: 1:5000), anti-MMP2 (bs-0412R, Rabbit, Bioss, 1:500, secondary antibody: 1:5000), anti-CoL1 (bs-10423R, Rabbit, Bioss, 1:500, secondary antibody: 1:5000), and anti-GAPDH (60004-1-lg, Mouse, Proteintech, 120,000, secondary antibody, 1:5000).

Techniques:

Journal: ACS Omega

Article Title: Mechanisms and Molecular Targets of BuShenHuoXue Formula for Osteoarthritis

doi: 10.1021/acsomega.1c07270

Figure Lengend Snippet: PCR Primer Sequences

Article Snippet: The following antibodies and corresponding dilution ratios were used: anti-SELE (bs-1273R, Rabbit, Bioss, 1:500, secondary antibody: 1:5000), anti-MMP2 (bs-0412R, Rabbit, Bioss, 1:500, secondary antibody: 1:5000), anti-CoL1 (bs-10423R, Rabbit, Bioss, 1:500, secondary antibody: 1:5000), and anti-GAPDH (60004-1-lg, Mouse, Proteintech, 120,000, secondary antibody, 1:5000).

Techniques: Sequencing