ADAMTS12 promotes fibrosis by restructuring extracellular matrix to enable activation of injury-responsive fibroblasts

Description

Methods

Inducible Fate Tracing - Microarray

Mice

Gli1CreERt2 (JAX stock #007913) and Rosa26tdTomato (JAX stock #007909) were purchased from Jackson Laboratories (Bar Harbor, ME, USA).

Unilateral Ureteral Obstruction

For inducible fate tracking, Gli1CreER;tdTomato mice (8 weeks old, n=3, 3m) received tamoxifen three times by gavage (10 mg p.o.) followed by a washout period of 21 days. Gli1CreER;tdTomato as well as Adamts12—/— (n=6, 2f, 4m, 20-24 weeks) and WT (n=7, 4f, 3m, 18-24 weeks) mice underwent Unilateral Ureter Obstruction (UUO) and contralateral sham surgery as previously described. In short, mice were anesthetized with 120 mg/kg body weight Ketamine and 16 mg/kg body weight Xylazine. After induction of narcosis, the left flank was incised and the left ureter ligated at the level of the lower pole with two 5.0 sutures (Mersilene). For sham surgery, an isolated flank incision was placed on the contralateral right flank of the same animal. For analgesia, 200 mg/kg body weight in 100 µl NaCl solution was administered subcutaneously 30 minutes prior to surgery. After surgery, metamizole (1.25 mg/ml) was added to the drinking water in combination with 1% sucrose for 72 hours. Mice were subsequently sacrificed via cardiac puncture in ketamine/xylazine narcosis. A small incision was placed in the right ventricle, after which the left ventricle was perfused with 20 ml of 4°C PBS to remove residual blood from the vasculature.

Fluorescence-activated cell sorting (FACS) and Affymetrix Microarray

Kidneys were minced into small, approx. 1 mm2 slices and transferred to a digestion solution containing 25 μg/ml Liberase TL (Roche) and 50 μg/ml DNase (Sigma) in RPMI (Gibco) in a C-tube (Miltenyi Biotec). Tissue was processed on a gentle-MACS (Miltenyi Biotec) using the program Spleen 4 and subsequently digested for 30 min at 37°C while shaking at 300 RPM before processing again on the gentle MACS using the Spleen 4 program. The resulting suspension was passed through a 70 μm cell strainer (Falcon), washed with 45 ml of cold PBS, and centrifuged for 5 minutes at 500 G at 4°C. Cells were counted using a hemocytometer with trypan blue staining. Overall viability by this method was greater than 80%. Isolated cells were resuspended in FACS-Buffer (1% fetal bovine serum in PBS) on ice at a final concentration of 1x107 cells/ml and filtered using a 40 µm cell strainer (Falcon). Live, single cells were isolated by FACS sorting using a FACS Aria II instrument (Becton Dickinson, Basel, Switzerland) and gating for Gli1-tdTomato positive, DAPI negative cells. On average, it took 5-6 hours from obtaining the biopsies to preparing the single-cell suspensions. After Gli1 cell isolation a Affymetrix GeneChip™ Mouse Genome 430 2.0 Array was performed according to the manufacturer's instructions.

Microarray analysis

Microarray gene expression was quantified using the R package affy (v.1.72) aligning probes to the mouse genome Mouse4302.db and normalizing gene expression using the Robust-Multichip Average (RMA-) function. After principal component analysis, the R package limma (v.3.44.1) was used to test differential gene expression between UUO and sham conditions using the RunLimma function. In case that two or more microarray probes mapped to the same gene, duplicate genes were removed. PROGENy and DoRothEA transcription factor analyses were performed as described below.

Methods

MI Visium

Mice

Adamts12—/— mice were developed by C. Lopez-Otin. Genotyping of all mice was performed by PCR. All animal experiments and procedures were approved by regional authorities (LANUV-NRW, Germany). 1 to 5 mice were housed together with unlimited admission to water and food on a 12-hour light/dark cycle, at 20°C under specific-pathogen-free conditions at RWTH Aachen University. Adamts12—/— mice were normally fertile with normal lifespans and no overt phenotype.

Myocardial infarction

To compare cardiac fibrosis in WT vs Adamts12—/— mice 56 days after MI, 11 to 17 week old Adamts12—/— underwent left anterior descending coronary artery ligation (n=11, 6f, 5m) or sham surgery (n=9, 4f, 5m). As specifically requested by regional authorities (LANUV-NRW, Germany), we had to use the same control group of WT sham and MI operated mice, which we previously operated to compare fibrosis in WT vs Cxcl4—/— animals. For Picro-Sirius Red stainings, heart sections of WT and Adamts12—/— were stained anew as described below. For echocardiography, data of WT mice was reanalyzed together with echocardiographic data of Adamts12—/— mice by a blinded trained professional as described below. For Visium spatial transcriptomic experiments 8 weeks old Adamts12—/— (n=4, 4f) and WT (n=4, 4f) mice were used. Mice were anesthetized using 2-2.5% isoflurane. For analgesia, metamizole (200 mg/kg body weight in 100 µl NaCl solution) was administered subcutaneously 30 minutes prior to surgery in addition to local analgesia by subcutaneous and intercostal injection of Bupivacaine (2,5 mg/kg BW). After induction of narcosis, mice were intubated and ventilated with oxygen by mouse respirator (Harvard Apparatus). Left thoracotomy was performed and MI was induced by ligature of the left anterior descending artery using 0/7 silk (Seraflex, IO0517IZ). For sham surgery an isolated left thoracotomy without left anterior descending coronary artery ligation was performed. After the ligation the ribs, muscle layer and skin incision were closed via suture and Metamizole was administered for three days via drinking water (1.25% mg/ml, 1% sucrose). Mice were sacrificed at 7 or 56 days after MI as described above.

Spatial gene expression assay

Spatial gene expression profiling (Visium, 10X Genomics, PN-1000187) including library construction of 10 µm OCT-embedded murine heart sections were performed according to the manufacturer's instructions. Optimal lysis time was determined around 18 minutes. Brightfield images were taken using a FRITZ scanner microscope. After library construction, libraries were sequenced on a NovaSeq 6000 System (Illumina) as recommended by 10X Genomics.

Methods

Bulk RNA Sequencing - WT vs KO, KO vs Act vs Inact

Bulk RNA Library Construction

Cultured cells were lysed, homogenized using QIAshredder spin columns (79656, Qiagen) and RNA was isolated as described above. Total RNA (200 ng) was reverse transcribed with High-Capacity cDNA Reverse Transcription Kit (4368813, Applied Biosystems). For cDNA and library construction of WT vs ADAMTS12-KO libraries we used the MGIEasy RNA Library Prep Set (MGI, 1000006384) in combination with the MGIEasy rRNA Depletion Kit (MGI, 1000005953) according to the manufacturer's instructions. After library construction and quality control using the AgilentTapeStation System, libraries were sequenced on a DnbSeq-G400 system, targeting a read depth of 25,000,000 reads/library. For cDNA and library construction of ADAMTS12-KO vs active or inactive ADAMTS12 expressing samples we used the NEBNext Ultra II Directional RNA Library Prep Kit (NEB, E7760L) according to the manufacturer's instructions. After quality control on the AgilentTapeStation System, samples were sequenced on an Illumina NovaSeq system, targeting a read depth of 25,000,000 reads/library.

Bulk RNA Analysis

Bulk RNA data was preprocessed as recommended by the nf-core nextflow pipeline (version 21.04.1) using nf-core/rnaseq (version 3.1), star (version 2.7.9a) for read alignment, salmon (version 1.5.0) for read quantification, trimgalore (version 0.6.6) for read trimming, and gencode (version 38) for gene annotation. For differential gene expression analysis, lowly expressed genes, “rRNA” ,”tRNA” and “mtRNA” were filtered out. Subsequently, differentially expressed genes were calculated using DESeq2 (version 1.320.