Perivascular Fibro-Adipogenic Progenitor Tracing during Post-Traumatic Osteoarthritis
2020; Elsevier BV; Volume: 190; Issue: 9 Linguagem: Inglês
10.1016/j.ajpath.2020.05.017
ISSN1525-2191
AutoresTakashi Sono, Ching-Yun Hsu, Yiyun Wang, Jiajia Xu, Masnsen Cherief, Simone Marini, Amanda K. Huber, Sarah Miller, Bruno Péault, Benjamin Lévi, Aaron W. James,
Tópico(s)Mesenchymal stem cell research
ResumoPerivascular mural cells surround capillaries and microvessels and have diverse regenerative or fibrotic functions after tissue injury. Subsynovial fibrosis is a well-known pathologic feature of osteoarthritis, yet transgenic animals for use in visualizing perivascular cell contribution to fibrosis during arthritic changes have not been developed. Here, inducible Pdgfra-CreERT2 reporter mice were subjected to joint-destabilization surgery to induce arthritic changes, and cell lineage was traced over an 8-week period with a focus on the joint-associated fat pad. Results showed that, at baseline, inducible Pdgfra reporter activity highlighted adventitial and, to a lesser extent, pericytic cells within the infrapatellar fat pad. Joint-destabilization surgery was associated with marked fibrosis of the infrapatellar fat pad, accompanied by an expansion of perivascular Pdgfra-expressing cellular descendants, many of which adopted α-smooth muscle actin expression. Gene expression analysis of microdissected infrapatellar fat pad confirmed enrichment in membrane-bound green fluorescent protein/Pdgfra-expressing cells, along with a gene signature that corresponded with injury-associated fibro-adipogenic progenitors. Our results highlight dynamic changes in joint-associated perivascular fibro-adipogenic progenitors during osteoarthritis. Perivascular mural cells surround capillaries and microvessels and have diverse regenerative or fibrotic functions after tissue injury. Subsynovial fibrosis is a well-known pathologic feature of osteoarthritis, yet transgenic animals for use in visualizing perivascular cell contribution to fibrosis during arthritic changes have not been developed. Here, inducible Pdgfra-CreERT2 reporter mice were subjected to joint-destabilization surgery to induce arthritic changes, and cell lineage was traced over an 8-week period with a focus on the joint-associated fat pad. Results showed that, at baseline, inducible Pdgfra reporter activity highlighted adventitial and, to a lesser extent, pericytic cells within the infrapatellar fat pad. Joint-destabilization surgery was associated with marked fibrosis of the infrapatellar fat pad, accompanied by an expansion of perivascular Pdgfra-expressing cellular descendants, many of which adopted α-smooth muscle actin expression. Gene expression analysis of microdissected infrapatellar fat pad confirmed enrichment in membrane-bound green fluorescent protein/Pdgfra-expressing cells, along with a gene signature that corresponded with injury-associated fibro-adipogenic progenitors. Our results highlight dynamic changes in joint-associated perivascular fibro-adipogenic progenitors during osteoarthritis. Perivascular mural cells surround capillaries and microvessels, and have various regenerative or fibrotic functions after tissue injury. In addition to regulation of angiogenesis,1Lindblom P. Gerhardt H. Liebner S. Abramsson A. Enge M. Hellstrom M. Backstrom G. Fredriksson S. Landegren U. Nystrom H.C. Bergstrom G. Dejana E. Ostman A. Lindahl P. Betsholtz C. Endothelial PDGF-B retention is required for proper investment of pericytes in the microvessel wall.Genes Dev. 2003; 17: 1835-1840Crossref PubMed Scopus (452) Google Scholar mural cells demonstrate tissue-intrinsic regenerative properties,2Volz K.S. Jacobs A.H. Chen H.I. Poduri A. McKay A.S. Riordan D.P. Kofler N. Kitajewski J. Weissman I. Red-Horse K. Pericytes are progenitors for coronary artery smooth muscle.Elife. 2015; 4: e10036Crossref PubMed Scopus (13) Google Scholar,3Crisan M. Yap S. Casteilla L. Chen C.W. Corselli M. Park T.S. Andriolo G. Sun B. Zheng B. Zhang L. Norotte C. Teng P.N. Traas J. Schugar R. Deasy B.M. Badylak S. Buhring H.J. Giacobino J.P. Lazzari L. Huard J. Peault B. A perivascular origin for mesenchymal stem cells in multiple human organs.Cell Stem Cell. 2008; 3: 301-313Abstract Full Text Full Text PDF PubMed Scopus (2761) Google Scholar but also contribute to organ fibrosis, such as in the kidney,4Kramann R. Schneider R.K. DiRocco D.P. Machado F. Fleig S. Bondzie P.A. Henderson J.M. Ebert B.L. Humphreys B.D. Perivascular Gli1+ progenitors are key contributors to injury-induced organ fibrosis.Cell Stem Cell. 2015; 16: 51-66Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar lung,5Hung C.F. Wilson C.L. Schnapp L.M. Pericytes in the lung.Adv Exp Med Biol. 2019; 1122: 41-58Crossref PubMed Scopus (10) Google Scholar,6Hung C. Linn G. Chow Y.H. Kobayashi A. Mittelsteadt K. Altemeier W.A. Gharib S.A. Schnapp L.M. Duffield J.S. Role of lung pericytes and resident fibroblasts in the pathogenesis of pulmonary fibrosis.Am J Respir Crit Care Med. 2013; 188: 820-830Crossref PubMed Scopus (211) Google Scholar liver,7Puche J.E. Saiman Y. 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Alphav integrins on mesenchymal cells regulate skeletal and cardiac muscle fibrosis.Nat Commun. 2017; 8: 1118Crossref PubMed Scopus (50) Google Scholar Indeed, various lineage-tracing strategies have been used for the identification of perivascular mural cells that contribute to organ fibrosis, including cells expressing FOXD110Humphreys B.D. Lin S.L. Kobayashi A. Hudson T.E. Nowlin B.T. Bonventre J.V. Valerius M.T. McMahon A.P. Duffield J.S. Fate tracing reveals the pericyte and not epithelial origin of myofibroblasts in kidney fibrosis.Am J Pathol. 2010; 176: 85-97Abstract Full Text Full Text PDF PubMed Scopus (988) Google Scholar and GLI1.4Kramann R. Schneider R.K. DiRocco D.P. Machado F. Fleig S. Bondzie P.A. Henderson J.M. Ebert B.L. Humphreys B.D. Perivascular Gli1+ progenitors are key contributors to injury-induced organ fibrosis.Cell Stem Cell. 2015; 16: 51-66Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar Thus, perivascular cells (or more likely subsets of mural cells within vessel walls) can demonstrate profibrotic features after tissue injury. Platelet-derived growth factor receptor α (PDGFRA) has been previously used for the identification of a bone marrow–resident skeletal progenitor cell population,11Houlihan D.D. Mabuchi Y. Morikawa S. Niibe K. Araki D. Suzuki S. Okano H. Matsuzaki Y. Isolation of mouse mesenchymal stem cells on the basis of expression of Sca-1 and PDGFR-alpha.Nat Protoc. 2012; 7: 2103-2111Crossref PubMed Scopus (170) Google Scholar or more generically as a marker of mesenchymal stem/stromal cells.11Houlihan D.D. Mabuchi Y. Morikawa S. Niibe K. Araki D. Suzuki S. Okano H. Matsuzaki Y. Isolation of mouse mesenchymal stem cells on the basis of expression of Sca-1 and PDGFR-alpha.Nat Protoc. 2012; 7: 2103-2111Crossref PubMed Scopus (170) Google Scholar PDGFRA-expressing progenitor cells within skeletal muscle are also commonly referred to as fibro-adipogenic progenitors (FAPs),12Arrighi N. Moratal C. Clement N. Giorgetti-Peraldi S. Peraldi P. Loubat A. Kurzenne J.Y. Dani C. Chopard A. Dechesne C.A. Characterization of adipocytes derived from fibro/adipogenic progenitors resident in human skeletal muscle.Cell Death Dis. 2015; 6: e1733Crossref PubMed Scopus (64) Google Scholar given their ability to give rise to fibroblasts and adipocytes after skeletal muscle injury.13Joe A.W. Yi L. Natarajan A. Le Grand F. So L. Wang J. Rudnicki M.A. Rossi F.M. Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis.Nat Cell Biol. 2010; 12: 153-163Crossref PubMed Scopus (833) Google Scholar In prior observations, Pdgfra-CreERT2;mT/mG reporter animals were useful for highlighting subsets of perivascular cells that would mobilize from the vascular wall during new tissue formation within s.c. adipose tissue.14Wang Y. Xu J. Meyers C.A. Gao Y. Tian Y. Broderick K. Peault B. James A.W. PDGFRalpha marks distinct perivascular populations with different osteogenic potential within adipose tissue.Stem Cells. 2020; 38: 276-290PubMed Google Scholar Yet, the role of perivascular PDGFRA-expressing cells and their descendants after tissue injury, such as during osteoarthritis, is not known. The morphologic features that accompany osteoarthritis after trauma are well catalogued. These features are best described within a rodent model of destabilization of the medial meniscus (DMM),15Glasson S.S. Askew R. Sheppard B. Carito B. Blanchet T. Ma H.L. Flannery C.R. Peluso D. Kanki K. Yang Z. Majumdar M.K. Morris E.A. Deletion of active ADAMTS5 prevents cartilage degradation in a murine model of osteoarthritis.Nature. 2005; 434: 644-648Crossref PubMed Scopus (918) Google Scholar,16Glasson S.S. Blanchet T.J. Morris E.A. The surgical destabilization of the medial meniscus (DMM) model of osteoarthritis in the 129/SvEv mouse.Osteoarthritis Cartilage. 2007; 15: 1061-1069Abstract Full Text Full Text PDF PubMed Scopus (673) Google Scholar but are broadly applicable to degenerative changes in osteoarthritis in humans.17Lorenz J. Grassel S. Experimental osteoarthritis models in mice.Methods Mol Biol. 2014; 1194: 401-419Crossref PubMed Scopus (43) Google Scholar Of the morphologic features, accumulation of collagenous extracellular matrix and fibrosis of the infrapatellar fat pad (IFP) are common findings.18Eymard F. Pigenet A. Citadelle D. Tordjman J. Foucher L. Rose C. Flouzat Lachaniette C.H. Rouault C. Clement K. Berenbaum F. Chevalier X. Houard X. Knee and hip intra-articular adipose tissues (IAATs) compared with autologous subcutaneous adipose tissue: a specific phenotype for a central player in osteoarthritis.Ann Rheum Dis. 2017; 76: 1142-1148Crossref PubMed Scopus (48) Google Scholar Diarthrodial joint–associated tissues, such as IFP, house multipotent perivascular cells,19Khan W.S. Tew S.R. Adesida A.B. Hardingham T.E. Human infrapatellar fat pad-derived stem cells express the pericyte marker 3G5 and show enhanced chondrogenesis after expansion in fibroblast growth factor-2.Arthritis Res Ther. 2008; 10: R74Crossref PubMed Scopus (86) Google Scholar,20Hindle P. Khan N. Biant L. Peault B. The infrapatellar fat pad as a source of perivascular stem cells with increased chondrogenic potential for regenerative medicine.Stem Cells Transl Med. 2017; 6: 77-87Crossref PubMed Scopus (46) Google Scholar presumably which also have a potential for fibrogenesis on injury. However, the role of perivascular FAPs in these histopathologic changes is entirely unknown. Here, we utilized transgenic Pdgfra-CreERT2;mT/mG reporter mice for documenting the kinetics and fate of Pdgfra-expressing cells within the knee joint after DMM surgery. All animal studies were performed with the approval of the institutional Animal Care and Use Committee at Johns Hopkins University (Baltimore, MD), and in compliance with all relevant ethics regulations. Pdgfra-CreERT2 mice were a kind gift from the Bergles Laboratory at Johns Hopkins, commercially available from The Jackson Laboratory (catalog number 018280; Bar Harbor, ME). mT/mG mice were purchased from The Jackson Laboratory (catalog number 007576). Pdgfra-CreERT2 and mT/mG mice were crossed to generate Pdgfra-CreERT2;mT/mG mice, which were used for all experiments. Tamoxifen (TM; product number T5648; Sigma-Aldrich, St. Louis, MO) was provided by i.p. injection as per previously validated protocols to mixed-sex, 7-week–old Pdgfra-CreERT2;mT/mG mice for 5 days consecutively (150 μg/g by weight).14Wang Y. Xu J. Meyers C.A. Gao Y. Tian Y. Broderick K. Peault B. James A.W. PDGFRalpha marks distinct perivascular populations with different osteogenic potential within adipose tissue.Stem Cells. 2020; 38: 276-290PubMed Google Scholar With this previously validated TM-injection schedule, >93.7% fidelity between reporter activity and immunolocalization was previously observed.21Kang S.H. Fukaya M. Yang J.K. Rothstein J.D. Bergles D.E. NG2+ CNS glial progenitors remain committed to the oligodendrocyte lineage in postnatal life and following neurodegeneration.Neuron. 2010; 68: 668-681Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar TM was dissolved in sunflower seed oil. Tail genomic DNA was used for genotyping. Essentially no recombination within the joint-associated tissues was observed with the injection of TM-free control vehicle. DMM or sham surgery was performed at the age of 10 weeks, in similarity to prior reports.16Glasson S.S. Blanchet T.J. Morris E.A. The surgical destabilization of the medial meniscus (DMM) model of osteoarthritis in the 129/SvEv mouse.Osteoarthritis Cartilage. 2007; 15: 1061-1069Abstract Full Text Full Text PDF PubMed Scopus (673) Google Scholar Briefly, left hindlimbs were disinfected with povidone–iodine and 70% ethanol, and suspended-release buprenorphine (1 mg/kg) was injected s.c. A 1-cm longitudinal incision was made on the medial aspect of the knee joint in mice under general anesthesia, using 2% isoflurane. Blunt dissection of the joint capsule along the medial side of the patellar ligament was performed to expose the medial meniscotibial ligament. The IFP was exposed, displaced laterally, and the medial meniscotibial ligament was transected using a number 11–blade scalpel to destabilize the medial meniscus. The medial joint capsule and the skin were next closed with 5-0 Prolene suture (Ethicon, Bridgewater, NJ). Sham surgery was performed with a similar surgical approach with visualization, but without transection of the medial meniscotibial ligament. In total, 15 mice were used for histologic examination (3 uninjured, 6 at 2 weeks after surgery, and 6 at 8 weeks after surgery), and 6 mice were used for gene expression experiments (3 sham operated, 3 DMM operated). Knee joints were dissected and imaged with the SkyScan1175 high-resolution micro computed tomography system (Bruker, Billerica, MA) at 65 kV and 153 μA with a 1.0-mm aluminum filter to obtain a 10-μm voxel size. Images were reconstructed using CT Vox Micro-CT Volume Rendering software version 3.2 (Bruker). Knee joint specimens were fixed in 4% paraformaldehyde for 24 hours, decalcified with 14% EDTA for 14 days, and embedded in OCT compound. Sagittal sections of the stifle joint were prepared at 6-μm thickness with a Cryofilm type 3c (Section-Lab, Hiroshima, Japan). As a comparison tissue, inguinal fat pads were dissected, embedded in OCT compound (Sakura, Torrance, CA), and cryosectioned at 30-μm thickness. Routine hematoxylin and eosin, Masson's trichrome, Safranin O/Fast Green, and Picro Sirius Red staining were performed, with methods adopted from past work.22Levi B. James A.W. Nelson E.R. Peng M. Wan D.C. Commons G.W. Lee M. Wu B. Longaker M.T. Acute skeletal injury is necessary for human adipose-derived stromal cell-mediated calvarial regeneration.Plast Reconstr Surg. 2011; 127: 1118-1129Crossref PubMed Scopus (31) Google Scholar, 23Lee S. Shen J. Pan H.C. Shrestha S. Asatrian G. Nguyen A. Meyers C. Nguyen V. Lee M. Soo C. Ting K. James A.W. Calvarial defect healing induced by small molecule smoothened agonist.Tissue Eng Part A. 2016; 22: 1357-1366Crossref PubMed Scopus (13) Google Scholar, 24Siu R.K. Zara J.N. Hou Y. James A.W. Kwak J. Zhang X. Ting K. Wu B.M. Soo C. Lee M. NELL-1 promotes cartilage regeneration in an in vivo rabbit model.Tissue Eng Part A. 2012; 18: 252-261Crossref PubMed Scopus (39) Google Scholar For immunofluorescence immunohistochemistry analysis, sections were washed in 1× phosphate-buffered saline, blocked in 5% normal goat serum (S-1000; Vector Laboratories, Burlingame, CA) for 30 minutes, and incubated with primary antibodies specific for CD31 (1:50; catalog number ab28364), α-smooth muscle actin (SMA) (1:400; catalog number ab7817), perilipin (Plin)-1 (1:500; catalog number ab3526), secreted frizzled-related protein (sFRP)-2 (1:100; catalog number ab92667), or macrophage migration inhibitory factor (Mif) (1:100; catalog number ab7207) (all, Abcam, Cambridge, MA) at 4°C overnight. Sections were then incubated with Alexa Fluor 647–conjugated secondary antibodies (1:200; catalog number ab150083 or ab150119; Abcam), and mounted with mounting medium containing DAPI (H-1500; Vector Laboratories). Bright field images were obtained on a Leica DM 6B microscope (Leica Biosystems, Wetzlar, Germany) or on a BX43 upright imaging system (Olympus, Tokyo, Japan). Immunofluorescence images were acquired on Leica DM 6B and LSM 780 FCS confocal microscopes (Carl Zeiss, Oberkochen, Germany). Tile scans from three to four samples per group were quantified by using ImageJ software version 1.49v (NIH, Bethesda, MD; http://imagej.nih.gov/ij) for reporter activity. Three frozen semiserial sagittal sections with 36-μm intervals were prepared from the medial compartment of each knee joint to represent the weight-bearing area of the distal femur and proximal tibia.25Yahara Y. Takemori H. Okada M. Kosai A. Yamashita A. Kobayashi T. Fujita K. Itoh Y. Nakamura M. Fuchino H. Kawahara N. Fukui N. Watanabe A. Kimura T. Tsumaki N. Pterosin B prevents chondrocyte hypertrophy and osteoarthritis in mice by inhibiting Sik3.Nat Commun. 2016; 7: 10959Crossref PubMed Scopus (40) Google Scholar Safranin O/Fast Green–stained sections were analyzed for cartilage injury using the Osteoarthritis Research Society International (OARSI) scoring system.26Pritzker K.P. Gay S. Jimenez S.A. Ostergaard K. Pelletier J.P. Revell P.A. Salter D. van den Berg W.B. Osteoarthritis cartilage histopathology: grading and staging.Osteoarthritis Cartilage. 2006; 14: 13-29Abstract Full Text Full Text PDF PubMed Scopus (1230) Google Scholar A range of 0 to 24 was determined according to the following formula: score = grade (G1–G6) × stage (S1–S4). The mean scores from both the femur and the tibia were summed. Sagittal sections at the level of the posterior cruciate ligament were stained with hematoxylin and eosin. Synovitis was scored using previously published synovitis scoring criteria,27Lewis J.S. Hembree W.C. Furman B.D. Tippets L. Cattel D. Huebner J.L. Little D. DeFrate L.E. Kraus V.B. Guilak F. Olson S.A. Acute joint pathology and synovial inflammation is associated with increased intra-articular fracture severity in the mouse knee.Osteoarthritis Cartilage. 2011; 19: 864-873Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar ranging from 0 (minimal inflammation) to 6 (maximal synovitis). Masson's trichrome–stained sagittal sections at the level of the posterior cruciate ligament were analyzed for fibrosis of the IFP. Semiquantitative evaluation was performed according to a method previously described.28Inomata K. Tsuji K. Onuma H. Hoshino T. Udo M. Akiyama M. Nakagawa Y. Katagiri H. Miyatake K. Sekiya I. Muneta T. Koga H. Time course analyses of structural changes in the infrapatellar fat pad and synovial membrane during inflammation-induced persistent pain development in rat knee joint.BMC Musculoskelet Disord. 2019; 20: 8Crossref PubMed Scopus (10) Google Scholar Briefly, 0 (fibrosis is 60%) indicates extensive. To analyze gene expression, IFPs were collected under a dissecting microscope at 2 weeks after sham or DMM surgery (n = 3 animals per condition). Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA) and the RNeasy Plus mini kit (Qiagen, Germantown, MD). Fifty nanograms of total RNA was used for synthesizing cDNA with the iScript cDNA synthesis kit (Bio-Rad, Philadelphia, PA). Real-time quantitative PCR was performed using QuantStudio 5 (Thermo Fisher Scientific) with PowerUp SYBR Green master mix (Thermo Fisher Scientific). The sequences of the primers used for PCR are shown in Table 1.Table 1Real-Time Quantitative RT-PCR Primer SequencesGeneForwardReverseActa25′-AGCCATCTTTCATTGGGATGG-3′5′-CCCCTGACAGGACGTTGTTA-3′Ccl25′-TTAAAAACCTGGATCGGAACCAA-3′5′-GCATTAGCTTCAGATTTACGGGT-3′Ccl75′-GTGTCCCTGGGAAGCTGTTA-3′5′-AGAAAGAACAGCGGTGAGGA-3′Cebpa5′-GGAACTTGAAGCACAATCGATC-3′5′-TGGTTTAGCATAGACGTGCACA-3′Col1a15′-TGTGTGCGATGACGTGCAAT-3′5′-CGGTCCCTCGACTCCTACA-3′Col1a25′-CCAGCGAAGAACTCATACAGC-3′5′-GGACACCCCTTCTACGTTGT-3′Col3a15′-ACGTAAGCACTGGTGGACAGA-3′5′-GAGGGCCATAGCTGAACTGA-3′Col6a15′-GCTGCTACAAGCCTGCTGGG-3′5′-GGCCCCATAAGGTTTCAGCC-3′Ctgf (Ccn2)5′-TCCACCCGAGTTACCAA-3′5′-TTAGGTGTCCGGATGC-3′Cxcl15′-CAAGAACATCCAGAGCTTGAAGGT-3′5′-GTGGCTATGACTTCGGTTTGG-3′Eno15′-AGCGATCCTACTGCCAGAAAT-3′5′-GATCGACCTCAACAGTGGGA-3′Gapdh5′-GACTTCAACAGCAACTCCCAC-3′5′-TCCACCACCCTGTTGCTG-3′Ier35′-GAGCGGGCCGTGGTGTC-3′5′-CTTGGCAATGTTGGGTTCCTC-3′Mgfp5′-CAACCACTACCTGAGCACCC-3′5′-GTCCATGCCGAGAGTGATCC-3′Mif5′-GCCAGAGGGGTTTCTGTCG-3′5′-GTTCGTGCCGCTAAAAGTCA-3′Pdgfrb5′-AGGACAACCGTACCTTGGGTGACT-3′5′-CAGTTCTGACACGTACCGGGTCTC-3′Pparg5′-AGGCCGAGAAGGAGAAGCTGTTG-3′5′-TGGCCACCTCTTTGCTCTGCTC-3′Ptx35′-CGCTGTGCTGGAGGAACT-3′5′-ATTGCTGTTTCACAACCTGC-3′Serpine15′-TTCAGCCCTTGCTTGCCTC-3′5′-ACACTTTTACTCCGAAGTCGGT-3′Sfrp25′-AGCCTGAGAATCGGCATCTA-3′5′-TATTTGAGGGCATCATGCAA-3′Timp15′-AGCCTCTGTGGATATGCCC-3′5′-TCAGAGTACGCCAGGGAACC-3′ Open table in a new tab To identify the genes involved in FAP-mediated fibrosis, a single-cell RNA sequencing data set derived from a rodent model of severe skeletal muscle fibrosis was evaluated. Briefly, fibrosis was induced by a polytrauma, in which an ischemia-generating injury (clamping of the left femoral artery), occluding bloodfor 3 hours (ischemia-reperfusion injury) was combined with an injection of 10 μL of cardiotoxin (3 mg/mL) into the left tibialis anterior, inducing local injury (ischemia-reperfusion injury/cardiotoxin). Baseline uninjured muscle (day 0) and post-trauma tibialis anterior muscles (day 3) were harvested from six ischemia-reperfusion injury/cardiotoxin-injured mice and eight uninjured mice. Tibialis anterior muscles were harvested and digested for 20 minutes at 37°C under constant agitation with 1640 RPMI media containing 750 U/mL of type 1 collagenase and 7 U/mL of Dispase II (Gibco, Gaithersburg, MD). These were quenched with 2% fetal bovine serum in phosphate-buffered saline and filtered through 40-μm sterile strainers. Cells were resuspended at a concentration of ∼1000 cells/μL. Single-cell 3′ library generation was performed on the 10× Genomics Chromium Controller following the manufacturer's protocol for the v2 reagent kit (10x Genomics, Pleasanton, CA). Samples were sequenced using the HiSeq 4000 system (Illumina, San Diego, CA). After sequencing, Cell Ranger Single Cell software suite 1.3 (10x Genomics) was used for performing sample de-multiplexing, barcode processing, and single-cell gene counting [alignment, barcoding, and unique molecular identifier (UMI) count] at the University of Michigan Biomedical Core Facilities DNA Sequencing Core (Ann Arbor, MI). A total of approximately 200 million reads were generated from the 10x Genomics sequencing analysis for each of the replicates (injured, three replicates, two animals each; uninjured, two replicates, four animals each). The sequencing data were first preprocessed using Cell Ranger and aligned to mm10 genome. For quality control, genes expressed in <10 cells, and cells expressing 20% mitochondrial UMI content, or UMI content >60,000 were filtered out. Replicates from the same group were pooled together for downstream analysis. Downstream analysis steps were performed using the Seurat version 2.3.0 pipeline (Sajita Lab, New York, NY). The downstream analysis steps for each sample type included normalization, scaling, dimensionality reduction (principal component analysis and t-distributed stochastic neighbor embedding), unsupervised clustering, cluster consolidation via centroid rank correlation analysis, and discovery of differentially expressed cluster-specific markers. The presence of replicate batch effect was ruled out by visual inspection of the contribution of each replicate to the principal component analysis and t-distributed stochastic neighbor embedding projections of the group. The clustering procedure followed two steps. Provisional clusters were assigned via unsupervised clustering (Seurat FindClusters; Louvain algorithm, k = 30, resolution = 0.4). This procedure led to eight provisional clusters in the injured group, and nine provisional clusters in the uninjured group. Provisional clusters were aligned according to the rank correlation of their centroids, measured on the gene set derived from the intersection of the genes for each set (12,619 genes). Consolidated clusters were obtained by aggregating similar clusters based on centroid rank correlation analysis, and their cell type was labeled according to their distinguishing markers. Pdgfra+ FAPs were selected from both groups, and their gene expression levels were compared for the finding of distinguishing markers. One-way analysis of variance tests followed by Tukey-Kramer tests were used for analysis of the results of each evaluation, except for gene expression results. The Dunnett test was used for analysis of gene expression results. All statistical analyses were performed using Prism software version 8 (GraphPad Software, San Diego, CA) and JMP software version 13 (SAS Institute Inc., Cary, NC). P < 0.05 was considered statistically significant. The distribution of Pdgfra-expressing cells was first examined within the IFP using Pdgfra-CreERT2;mT/mG reporter animals (Figure 1). For comparison, s.c. fat was also examined, which was previously described to contain two perivascular domains of Pdgfra reporter activity.14Wang Y. Xu J. Meyers C.A. Gao Y. Tian Y. Broderick K. Peault B. James A.W. PDGFRalpha marks distinct perivascular populations with different osteogenic potential within adipose tissue.Stem Cells. 2020; 38: 276-290PubMed Google Scholar For this experiment, TM was administered to Pdgfra-CreERT2;mT/mG transgenic mice for 5 consecutive days at 7 weeks of age (Figure 1A). First, it was confirmed that s.c. (inguinal) fat demonstrated perivascular Pdgfra reporter activity (Figure 1, B–D), primarily within the tunica adventitia of larger blood vessels (Figure 1C). A second, and more restricted, domain of perivascular Pdgfra reporter activity was also observed within a pericytic location in the tunica intima (Figure 1D). Similar perivascular distributions of Pdgfra reporter activity were found within the IFP of the knee joint (Figure 1, E–G). Here, Pdgfra reporter was again most common in the tunica adventitia of larger microvessels (Figure 1F), while some cells within a pericytic location also demonstrated reporter activity (Figure 1G). Thus, Pdgfra reporter activity within intracapsular fat pads demonstrates perivascular domains similar to those of s.c. fat.14Wang Y. Xu J. Meyers C.A. Gao Y. Tian Y. Broderick K. Peault B. James A.W. PDGFRalpha marks distinct perivascular populations with different osteogenic potential within adipose tissue.Stem Cells. 2020; 38: 276-290PubMed Google Scholar Pdgfra reporter animals underwent degenerative changes after DMM surgery, as expected (Figure 2). In these experiments, TM was administered to Pdgfra-CreERT2;mT/mG transgenic mice for 5 consecutive days at 7 weeks of age; mice were subjected to DMM or sham surgery at 10 weeks of age and analyzed at 2 and 8 weeks thereafter (Figure 2A). An uninjured group was also made to examine the baseline joint state at 10 weeks of age. Micro computed tomography of the left knee joint confirmed destabilization of the medial meniscus among the DMM treatment groups at 2 and 8 weeks after surgery (Figure 2B). Sagittal histologic sections of the joint were next stained with Safranin O/Fast Green (Figure 2, C and D). Results confirmed a significant and progressive degeneration of the anterior horn of the medial meniscus (Figure 2C) and loss of Safranin O staining among DMM animals (Figure 2D), but not sham-operated or uninjured animals (Figure 2, C and D). These findings were most notable within the proximal tibial articular cartilage, but were also evident within the distal femoral cartilage. Degenerative changes were further confirmed using the OARSI scoring system, which showed a progressive increase in scores across 2 and 8 weeks among DMM-treated animals (mean OARSI scores, 4.8 and 11.1 at 2 and 8 weeks after DMM, respectively) (Figure 2E). Having confirmed osteoarthritic changes after DMM, this study next examined in detail the histologic changes within IFP after either sham or DMM surgery. For this purpose, sagittal sections of the knee joint were obtained at the level of the posterior cruciate ligament (Figure 3) and stained with hematoxylin and eosin (Figure 3, A and B), Masson's trichrome (Figure 3C), and Picro Sirius Red (Figure 3D). Results showed marked fibrosis within the IFP as early as 2 weeks after DMM (Figure 3, C and D). In comparison, little histologic change within the IFP was observed in sham-operated animals. Inflammatory changes within the IFP and overlying synovium were next quantified using a semiquantitative scoring system (Figure 3E). Synovitis score was highest at 2 weeks after DMM and remained elevated at 8 weeks after DMM (mean synovitis scores, 5.0 and 4.3 at 2 and 8 weeks after DMM, respectively). Next, fibrosis was scored to quantify the qualitative impression of increased IFP fibrosis after joint destabilization (Figure 3F). Fibrosis score was significantly elevated among the DMM-treated groups only, observed at both 2 and 8 weeks after surgery (mean fibrosis scores, 2.7 and 2.0 at 2 and 8 weeks after DMM, respectivel
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