Journal: Journal of Biological Chemistry

Article Title: Fibronectin Binds and Enhances the Activity of Bone Morphogenetic Protein 1

doi: 10.1074/jbc.m109.024125

Figure Lengend Snippet: FIGURE 1. cFN and pFN bind BMP1. A, 80 ng of FLAG-tagged BMP1 was preincubated overnight with an equimolar amount of cFN or pFN. The com- plex was then immunoprecipitated (IP) with antisera raised against the N-ter- minal 70-kDa FN fragment (anti-70K), which recognizes both cFN and pFN. Immunoblotting (IB) was performed using either monoclonal anti-FLAG or polyclonal anti-70K antibody. B, BMP1 binding of cFN and pFN in ELISA. BMP1 at 3.4, 11.4, 34.1, 113.6, 340.6, and 1136.4 nM was incubated with cFN or pFN adsorbed to microtiter wells, as described under “Experimental Procedures.” The calculated KD values for BMP1 binding to cFN and pFN, as determined by Scatchard-typeplots,were11020and12030nM,respectively.Valuesare expressed as the mean S.D. of three experiments.

Article Snippet: Coverslips were then blocked with 0.1% BSA-PBS for 30 min followed by the addition of goat polyclonal antibody against BMP1 (R&D Systems) in 1% BSA-PBS (1:50) and incubation for 1 h at room temperature.

Techniques: Immunoprecipitation, Western Blot, Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Journal of Biological Chemistry

Article Title: Fibronectin Binds and Enhances the Activity of Bone Morphogenetic Protein 1

doi: 10.1074/jbc.m109.024125

Figure Lengend Snippet: FIGURE 3. FN binds BMP1 at multiple sites. FLAG-tagged BMP1 was preincubated overnight with various truncated forms of FN followed by immunoprecipitation (IP). A, immunoblots (IB) show that immunopre- cipitation of either the FN N-terminal 70-kDa fragment (70K) or the FN C-terminal fragment (III1-C) using anti-70K antibody or monoclonal LabMab anti-FN antibody, respectively, co-precipitated BMP1. B, immu- noblots show that immunoprecipitation of III1-C(EDA) or III1-C(EDA), with monoclonal LabMab anti- body, co-precipitated similar amounts of BMP1. C, immunoblots show that immunoprecipitation of III1–14 with monoclonal LabMab antibody co-precipitated BMP1. D, a schematic is shown of the protein domain structure of FN and of truncated forms of FN used in this study. FN types I, II, and III repeats are shown. Alternatively spliced regions EDA, EDB, and V sequences are indicated, as are linker sequences (horizontal lines).

Article Snippet: Coverslips were then blocked with 0.1% BSA-PBS for 30 min followed by the addition of goat polyclonal antibody against BMP1 (R&D Systems) in 1% BSA-PBS (1:50) and incubation for 1 h at room temperature.

Techniques: Immunoprecipitation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Fibronectin Binds and Enhances the Activity of Bone Morphogenetic Protein 1

doi: 10.1074/jbc.m109.024125

Figure Lengend Snippet: FIGURE 2. BMP1 binds FN via its non-protease domain sequences. A, an immunoblot (IB) shows input amounts of cFN, FLAG-tagged versions of full-length BMP1, a form of BMP1 containing all domains except the protease domain (BMP1-D), and isolated BMP1 protease domain (BMP1-P). B, after overnight incuba- tion of BMP1-D or BMP1-P with cFN, an immunoblot shows that the immunoprecipitation (IP) of cFN using anti-70K antibody pulls down full-length BMP1 and BMP1-D but not BMP1-P.

Article Snippet: Coverslips were then blocked with 0.1% BSA-PBS for 30 min followed by the addition of goat polyclonal antibody against BMP1 (R&D Systems) in 1% BSA-PBS (1:50) and incubation for 1 h at room temperature.

Techniques: Western Blot, Isolation, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: Fibronectin Binds and Enhances the Activity of Bone Morphogenetic Protein 1

doi: 10.1074/jbc.m109.024125

Figure Lengend Snippet: FIGURE 4. FN enhances BMP1 proteolytic processing of probiglycan, pro- collagen type I, and Chordin in vitro. Thirty-minute in vitro cleavage assays of probiglycan (A) or 6-h cleavage assays of type I procollagen (B and C) or Chordin (D) were performed in cFN-coated wells, with BSA-coated wells used as controls (A–C) or in the presence or absence of soluble cFN (D). A, probig- lycan (ProBgn) and mature biglycan (Bgn) were detected by immunoblot (IB) using antibody (LF51) directed against sequences in the mature region of biglycan (20). B and C, procollagen, processing intermediates, and mature 1(I) chains were detected by immunoblot using C-telopeptide polyclonal antibody LF67 (20). Samples in B and C were run on 7.5 and 6.0% acrylamide SDS-PAGEgels,respectively,thelattertoseparatepC-1(I)andpN-1(I)proc- essing intermediates. In B, in which reactions went somewhat further than in C, mature 1(I) chains are clearly discernable. D, BMP1 processing of C-termi- nal FLAG-tagged Chordin was monitored by immunoblot using anti-FLAG antibody. BMP1 cleaves Chordin at both N- and C-terminal sites (18). Bands for full-length Chordin (chd) and for a processing intermediate cleaved at the N-terminal but not the C-terminal site (C-chd) are marked but were not well separated in the 15% gel. Upon cleavage by BMP1, both bands disappeared, with the appearance of the 15-kDa Chordin C-terminal cleavage product (denoted by an asterisk), indicating complete Chordin processing.

Article Snippet: Coverslips were then blocked with 0.1% BSA-PBS for 30 min followed by the addition of goat polyclonal antibody against BMP1 (R&D Systems) in 1% BSA-PBS (1:50) and incubation for 1 h at room temperature.

Techniques: In Vitro, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Fibronectin Binds and Enhances the Activity of Bone Morphogenetic Protein 1

doi: 10.1074/jbc.m109.024125

Figure Lengend Snippet: FIGURE 5. Immunofluorescent co-localization of BMP1 and FN in fibro- blast ECM. Paraformaldehyde-fixed cultures of human neonatal fibroblasts, either permeabilized or not permeabilized with Triton X-100, were immuno- stained with antibodies to FN (green) and BMP1 (red) and were stained with 4,6-diamidino-2-phenylindole (DAPI, blue) for localization of nuclear DNA. The yellow signal indicates areas in which green and red signals overlap and, thus, areas of co-localization between FN and BMP1.

Article Snippet: Coverslips were then blocked with 0.1% BSA-PBS for 30 min followed by the addition of goat polyclonal antibody against BMP1 (R&D Systems) in 1% BSA-PBS (1:50) and incubation for 1 h at room temperature.

Techniques: Staining

Journal: Journal of Biological Chemistry

Article Title: Fibronectin Binds and Enhances the Activity of Bone Morphogenetic Protein 1

doi: 10.1074/jbc.m109.024125

Figure Lengend Snippet: FIGURE 6. Detection of endogenous BMP1-FN complexes in cell culture. Conditioned medium from cultures of MG-63 cells pretreated 24 h with TGF1 was subjected to immunoprecipitation (IP) with anti-BMP1 antibody (lane labeled IP BMP1 in panel A), anti-FN antibody (lane labeled IP FN in panelB),orIgG(IPIgG-labeledlanesinbothpanelsAandB)followedbyimmu- noblotting(IB)withanti-FNoranti-BMP1antibody.Inputlanesrepresent30l conditioned media not subjected to immunoprecipitation.

Article Snippet: Coverslips were then blocked with 0.1% BSA-PBS for 30 min followed by the addition of goat polyclonal antibody against BMP1 (R&D Systems) in 1% BSA-PBS (1:50) and incubation for 1 h at room temperature.

Techniques: Cell Culture, Immunoprecipitation, Labeling

Journal: Journal of Biological Chemistry

Article Title: Fibronectin Binds and Enhances the Activity of Bone Morphogenetic Protein 1

doi: 10.1074/jbc.m109.024125

Figure Lengend Snippet: FIGURE 7. Reduced processing of endogenous BMP1 substrates in cul- turesofFN/MEFs.A,confluentFN/andFN/MEFswereincubatedfor 24 h in serum-free medium, and endogenous Chordin (chd) from the condi- tioned medium, concentrated by ethanol precipitation or with heparin- Sepharose, was probed via immunoblotting (IB) using anti-N-terminal-Chor- din antibodies. Staining of ethanol-precipitated media samples with antibodies to secreted proteins sFRP2 and BMP1 shows similar levels of these two secreted proteins in FN/ and FN/ MEF media, arguing against over- all differences in levels of secreted proteins despite different levels of detected Chordin. Staining of cell extracts with anti--tubulin antibody dem- onstrates that FN/ and FN/ MEF cultures contained similar numbers of cells despite different levels of detected Chordin from conditioned media. B, confluent FN/ and FN/ MEFs were treated for 24 h with 50 g/ml ascor- bate and then incubated for 24 h in serum-free medium containing 50 g/ml ascorbatefollowedbyharvestingofmediaandcelllayersamples,whichwere then assayed via Western blot using LF67 antibody directed against the C-te- lopeptide of the 1(I) collagen chain. Positions are shown for procollagen, pN1(I) chains ((pN), processing intermediates from which C-propeptides have been removed but which retain N-propeptides), pC1(I) chains ((pC), processing intermediates from which N-propeptides have been removed but which retain C-propeptides), and mature, fully processed, 1(I) collagen chains. Reduced processing of the C-propeptides of type I procollagen and decreased deposition of mature collagen were observed in FN/ MEF cul- turescomparedwithculturesofFN/MEFs,indicatingdramaticallyreduced pCP activity for BMP1-like proteinases. C, confluent FN/ and FN/ MEFs were incubated for 24 h in serum-free medium, and probiglycan processing was examined in conditioned medium by Western blot using antibody LF51 directed against sequences within mature biglycan (20). Pro and mature forms of biglycan are denoted in the figure as ProBgn and Bgn, respectively. Probiglycan is less processed in FN/ MEF cultures than in FN/ MEF cultures.

Article Snippet: Coverslips were then blocked with 0.1% BSA-PBS for 30 min followed by the addition of goat polyclonal antibody against BMP1 (R&D Systems) in 1% BSA-PBS (1:50) and incubation for 1 h at room temperature.

Techniques: Ethanol Precipitation, Western Blot, Staining, Incubation, Activity Assay

Journal: Nature Communications

Article Title: Bone morphogenetic protein 1.3 inhibition decreases scar formation and supports cardiomyocyte survival after myocardial infarction

doi: 10.1038/s41467-021-27622-9

Figure Lengend Snippet: a Quantification of BMP1.3 protein levels in blood of healthy individuals and patients affected by acute myocardial infarction (MI) within 12 h from hospitalization. b Quantification of the expression levels of BMP1.3 normalized over GAPDH in human myocardium of patients without overt cardiac disease (control) or affected by ischemic heart disease (ischemic). c Quantification of BMP1-3 protein levels in the blood of sham-operated or infarcted mice at 24 h after MI. d Quantification of the expression levels of Bmp1.3 normalized over 18 S in the myocardium of sham-operated or infarcted mice at 48 h after MI. e Quantification of the levels of Bmp1.3 mRNA normalized on Actb in different cardiac cell types at 48 h after MI. f Quantification of the ejection fraction in rats treated with isoproterenol (Ip), alone (control) or in combination with anti-BMP1.3 antibody (150 µg/kg). Values for sham animals are also shown for reference. g Quantification of the area covered by collagen in each section. h Representative images of heart sections stained with Sirius red to show collagen deposition upon administration of isoproterenol alone (control) or in combination with anti-BMP1.3 antibody. i Quantification of the area covered by collagen in sections of murine hearts sham-operated or injured by myocardial infarction (MI) and treated with the indicated dose of anti-BMP1.3 antibody (150 or 500 µg/kg). j Representative images of murine heart sections stained with Sirius red to show collagen deposition upon MI and treatment with the indicated dose of anti-BMP1.3 antibody. k Quantification of the ejection fraction at 7, 14, 30, and 60 days after MI. Data in a , b , c , d , e , f , g , i , and k are shown as mean ± s.e.m. Sample number is indicated inside or above each bar. Statistical significance was determined using unpaired, two-sided t -test in a – d , one-way ANOVA, followed by Tukey’s test in e , f , g , i , and two-way ANOVA in k followed by Tukey’s multiple comparison test. Scale bar in h , j is 200 µm and 1 mm, respectively. Source data are provided as a Source Data file.

Article Snippet: Plasma levels of BMP1.3 were measured using the ELISA Human BMP1 Matched Antibody Pair Kit (Cloud-Clone PSA653Hu01) and additional reagents supplied in Antibody Pairs Support Pack (Cloud-Clone IS077).

Techniques: Expressing, Staining

Journal: Nature Communications

Article Title: Bone morphogenetic protein 1.3 inhibition decreases scar formation and supports cardiomyocyte survival after myocardial infarction

doi: 10.1038/s41467-021-27622-9

Figure Lengend Snippet: a Representative images of heart sections from COLL-EGFP mice after MI (control) and after treatment with anti-BMP1.3 antibody (150 µg/kg). Nuclei were stained with DAPI. b Quantification of the EGFP + area normalized on DAPI + area. c Quantification of both EGFP + or RFP + pixels in cultured cardiac fibroblasts from COLL-EGFP/SMA-RFP mice untreated (control), treated with human recombinant BMP1.3 (rhBMP1.3, 0.1 µg/ml) or anti-BMP1.3 antibody (BMP1.3 Ab, 5 µg/ml). d Representative images of cultured cardiac fibroblast from COLL-EGFP/SMA-RFP mice treated as indicated. e Quantification of the expression levels of Col1a , Lox, Ctgf, Fn , and Tgf1b genes normalized to Gapdh in cultured cardiac fibroblasts treated as described. f Representative images from tissue section of infarct area of animals untreated (control) or treated with anti-BMP1.3 antibody (150 µg/kg) stained with anti-Tgfβ1, anti-phospho-Smad2 and anti-CTGF antibodies (brown) and hematoxylin (violet) to visualize nuclei. g Quantification of the expression levels of Col1a , Ctgf, Fn, Lox , and Tgf1b genes normalized to Gapdh in hearts, either untreated (control) or treated with anti-BMP1.3 antibody (150 µg/kg). h Representative images from tissue section of the heart of sham animals or animals subjected to MI, either in the absence of treament (control) or treated with anti-BMP1.3 antibody (150 µg/kg), stained with anti-Lox antibodies (brown) and hematoxylin (violet) to visualize nuclei. i Biochemical analysis of collagen crosslinking in scar tissue dissected from hearts of untreated rats (control) or rats treated with anti-BMP1.3 antibody (150 µg/kg). DHNL dihydroxynorleucine, DHLNL dihydroxylysinonorleucine, HLNL hydroxylysinonorleucine, Pyr pyridinoline, d-Pyr deoxypyridinoline, ALD total aldehyde, NON RED non reduced aldehyde. Data in b , c , e , g and i are shown as mean ± s.e.m. Sample number is indicated inside or above each bar. Statistical significance was determined using unpaired, two-sided t -test in b , e , g and i or one-way ANOVA followed by Tukey’s multiple comparison test in c . Scale bars in a , f and h is 100 µm, while in d is 500 µm. Source data are provided as a Source Data file.

Article Snippet: Plasma levels of BMP1.3 were measured using the ELISA Human BMP1 Matched Antibody Pair Kit (Cloud-Clone PSA653Hu01) and additional reagents supplied in Antibody Pairs Support Pack (Cloud-Clone IS077).

Techniques: Staining, Cell Culture, Recombinant, Expressing

Journal: Nature Communications

Article Title: Bone morphogenetic protein 1.3 inhibition decreases scar formation and supports cardiomyocyte survival after myocardial infarction

doi: 10.1038/s41467-021-27622-9

Figure Lengend Snippet: a Representative images of TUNEL and α-actinin staining on heart sections of mice untreated (sham) or injured by a MI in the absence of treatment (control) or after injection of anti-BMP1.3 antibody (150 µg/kg). Nuclei were stained with DAPI. b Quantification of dying cells in the infarcted area. c Quantification of TUNEL + nuclei in primary cardiomyocytes and cardiac fibroblasts. d Representative images of primary cardiac cells, either untreated (control) or treated with anti-BMP1.3 antibody (5 µg/ml), stained with TUNEL and anti-α-actinin antibody. Nuclei were stained with DAPI. e Representative images of primary cardiac fibroblasts and cardiomyocytes, cultured in hypoxic conditions, either in the absence (control) or in the presence of recombinant BMP1.3 (0.1 µg/ml), stained with TUNEL and anti-α-actinin antibody. Nuclei were stained with DAPI. f Quantification of TUNEL + nuclei in cardiomyocytes and cardiac fibroblasts. Data in b , c , and f are shown as mean ± s.e.m. Sample number is indicated inside or above each bar. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparison test in b , c and f . Scale bars in a , d and e indicate 100 µm. Source data are provided as a Source Data file.

Article Snippet: Plasma levels of BMP1.3 were measured using the ELISA Human BMP1 Matched Antibody Pair Kit (Cloud-Clone PSA653Hu01) and additional reagents supplied in Antibody Pairs Support Pack (Cloud-Clone IS077).

Techniques: TUNEL Assay, Staining, Injection, Cell Culture, Recombinant

Journal: Nature Communications

Article Title: Bone morphogenetic protein 1.3 inhibition decreases scar formation and supports cardiomyocyte survival after myocardial infarction

doi: 10.1038/s41467-021-27622-9

Figure Lengend Snippet: a Quantification of the expression levels of Bmp2 and Bmp5 in primary cardiac fibroblasts and cardiomyocytes cultured in hypoxic conditions in the absence of treatment (control) or treated with anti-BMP1.3 antibody (5 µg/ml), normalized to Gapdh . b Quantification of the expression levels of Bmp2 and Bmp5 in the heart of mice subjected to MI in the absence of treatment (control) or after injection of anti-BMP1.3 antibody (150 µg/kg). c Representative images from tissue section of infarct area of untreated rats (control) or treated with anti-BMP1.3 antibody (150 µg/kg) stained with either anti-BMP2 or anti-BMP5 antibody (brown) and hematoxylin (violet) to visualize nuclei. d Quantification of living cardiomyocytes cultured in hypoxic conditions expressed as number of cardiomyocytes per field and exposed to the indicated enriched supernatants collected from CHO transfected with control plasmid (pGI) or plasmid for the overexpression of BMP2 and/or BMP5. e Representative images of rat neonatal cardiomyocytes cultured in hypoxic conditions and exposed to the indicated treatments. Cardiomyocytes were stained with anti-α-actinin antibody and nuclei with DAPI. f Quantification of living cardiomyocytes cultured in hypoxic conditions and exposed to the indicated treatments. g Representative images of rat neonatal cardiomyocytes cultured in hypoxic conditions in the absence of treatment (−Ab) or treated with anti-BMP1.3 antibody (+Ab) and transfected with the indicated siRNAs (mock = scramble siBMP5). Data in a , b , d and f are shown as mean ± s.e.m. Sample number is indicated inside or above each bar. Statistical significance was determined using unpaired, two-sided t -test in a , b or one-way ANOVA followed by Dunnet’s in d or Tukey’s multiple comparison test in f . Scale bar in c , e and g is 100 µm. Source data are provided as a Source Data file.

Article Snippet: Plasma levels of BMP1.3 were measured using the ELISA Human BMP1 Matched Antibody Pair Kit (Cloud-Clone PSA653Hu01) and additional reagents supplied in Antibody Pairs Support Pack (Cloud-Clone IS077).

Techniques: Expressing, Cell Culture, Injection, Staining, Transfection, Plasmid Preparation, Over Expression

Journal: Nature Communications

Article Title: Bone morphogenetic protein 1.3 inhibition decreases scar formation and supports cardiomyocyte survival after myocardial infarction

doi: 10.1038/s41467-021-27622-9

Figure Lengend Snippet: The anti-BMP1.3 monoclonal antibody blocks the enzymatic activity of BMP1.3, exerting pleiotropic therapeutic effects after MI. On the one hand, BMP1.3 inhibition reduces collagen maturation. On the other hand, it results in TGFβ pathway inhibition, which in turns reduces myofibroblast activation, Lox expression and collagen cross-linking and increases the expression of cardioprotective BMPs.

Article Snippet: Plasma levels of BMP1.3 were measured using the ELISA Human BMP1 Matched Antibody Pair Kit (Cloud-Clone PSA653Hu01) and additional reagents supplied in Antibody Pairs Support Pack (Cloud-Clone IS077).

Techniques: Activity Assay, Inhibition, Activation Assay, Expressing