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• W3213680757 abstract "Collagen XII is a regulator of corneal stroma structure and function. The current study examined the role of collagen XII in regulating corneal stromal transforming growth factor (TGF)-β activation and latency. Specifically, with the use of conventional collagen XII null mouse model, the role of collagen XII in the regulation of TGF-β latency and activity in vivo was investigated. Functional quantification of latent TGF-β in stromal matrix was performed by using transformed mink lung reporter cells that produce luciferase as a function of active TGF-β. Col12a1 knockdown with shRNA was used to test the role of collagen XII in TGF-β activation. Col12a1–/– hypertrophic stromata were observed with keratocyte hyperplasia. Increased collagen fibril forward signal was found by second harmonic generation microscopy in the absence of collagen XII. Collagen XII regulated mRNA synthesis of Serpine1, Col1a1, and Col5a1 and deposition of collagens in the extracellular matrix. A functional plasminogen activator inhibitor luciferase assay showed that collagen XII is necessary for latent TGF-β storage in the extracellular matrix and that collagen XII down-regulates active TGF-β. Collagen XII dictates stromal structure and function by regulating TGF-β activity. A hypertrophic phenotype in Col12a1–/– corneal tissue can be explained by abnormal up-regulation of TGF-β activation and decreased latent storage. Collagen XII is a regulator of corneal stroma structure and function. The current study examined the role of collagen XII in regulating corneal stromal transforming growth factor (TGF)-β activation and latency. Specifically, with the use of conventional collagen XII null mouse model, the role of collagen XII in the regulation of TGF-β latency and activity in vivo was investigated. Functional quantification of latent TGF-β in stromal matrix was performed by using transformed mink lung reporter cells that produce luciferase as a function of active TGF-β. Col12a1 knockdown with shRNA was used to test the role of collagen XII in TGF-β activation. Col12a1–/– hypertrophic stromata were observed with keratocyte hyperplasia. Increased collagen fibril forward signal was found by second harmonic generation microscopy in the absence of collagen XII. Collagen XII regulated mRNA synthesis of Serpine1, Col1a1, and Col5a1 and deposition of collagens in the extracellular matrix. A functional plasminogen activator inhibitor luciferase assay showed that collagen XII is necessary for latent TGF-β storage in the extracellular matrix and that collagen XII down-regulates active TGF-β. Collagen XII dictates stromal structure and function by regulating TGF-β activity. A hypertrophic phenotype in Col12a1–/– corneal tissue can be explained by abnormal up-regulation of TGF-β activation and decreased latent storage. The corneal stroma has intrinsic properties that are essential for transparency, biomechanics, curvature, and avascularity. These intrinsic stromal properties are dependent on a highly organized and corneal-specific extracellular matrix and a unique population of well-interconnected cranial neural crest–derived cells unique to the stroma and known as keratocytes.1Poole C.A. Brookes N.H. Clover G.M. Keratocyte networks visualised in the living cornea using vital dyes.J Cell Sci. 1993; 106: 685-691Google Scholar, 2Chen S. Mienaltowski M.J. Birk D.E. Regulation of corneal stroma extracellular matrix assembly.Exp Eye Res. 2015; 133: 69-80Google Scholar, 3Espana E.M. Birk D.E. Composition, structure and function of the corneal stroma.Exp Eye Res. 2020; 198: 108137Google Scholar, 4Meek K.M. Knupp C. Corneal structure and transparency.Prog Retin Eye Res. 2015; 49: 1-16Google Scholar, 5Jester J.V. Moller-Pedersen T. Huang J. Sax C.M. Kays W.T. Cavangh H.D. Petroll W.M. Piatigorsky J. The cellular basis of corneal transparency: evidence for “corneal crystallins”.J Cell Sci. 1999; 112: 613-622Google Scholar, 6Quantock A.J. Young R.D. Development of the corneal stroma, and the collagen-proteoglycan associations that help define its structure and function.Dev Dyn. 2008; 237: 2607-2621Google Scholar A component of this corneal-specific extracellular matrix is fibril-associated collagens with interrupted triple helices that interact with collagen fibrils, as well as basement membranes, and regulate cell-cell communication and matrix organization.2Chen S. Mienaltowski M.J. Birk D.E. Regulation of corneal stroma extracellular matrix assembly.Exp Eye Res. 2015; 133: 69-80Google Scholar,7Hemmavanh C. Koch M. Birk D.E. Espana E.M. Abnormal corneal endothelial maturation in collagen XII and XIV null mice.Invest Ophthalmol Vis Sci. 2013; 54: 3297-3308Google Scholar Fibril-associated collagens with interrupted triple helices members, collagens XII and XIV, are known components of the stromal extracellular matrix.3Espana E.M. Birk D.E. Composition, structure and function of the corneal stroma.Exp Eye Res. 2020; 198: 108137Google Scholar,7Hemmavanh C. Koch M. Birk D.E. Espana E.M. Abnormal corneal endothelial maturation in collagen XII and XIV null mice.Invest Ophthalmol Vis Sci. 2013; 54: 3297-3308Google Scholar, 8Young B.B. Zhang G. Koch M. Birk D.E. The roles of types XII and XIV collagen in fibrillogenesis and matrix assembly in the developing cornea.J Cell Biochem. 2002; 87: 208-220Google Scholar, 9Massoudi D. Malecaze F. Soler V. Butterworth J. Erraud A. Fournie P. Koch M. Galiacy S.D. NC1 long and NC3 short splice variants of type XII collagen are overexpressed during corneal scarring.Invest Ophthalmol Vis Sci. 2012; 53: 7246-7256Google Scholar Collagen XII is a homotrimer composed of three collagen α1(XII) chains. It is a large protein with two major alternatively spliced variants, and the large variant has a glycosaminoglycan attachment site.9Massoudi D. Malecaze F. Soler V. Butterworth J. Erraud A. Fournie P. Koch M. Galiacy S.D. NC1 long and NC3 short splice variants of type XII collagen are overexpressed during corneal scarring.Invest Ophthalmol Vis Sci. 2012; 53: 7246-7256Google Scholar The function of collagen XII is poorly understood, but it is implicated in the regulation of tissue organization and function in different tissues.10Schonborn K. Willenborg S. Schulz J.N. Imhof T. Eming S.A. Quondamatteo F. Brinckmann J. Niehoff A. Paulsson M. Koch M. Eckes B. Krieg T. Role of collagen XII in skin homeostasis and repair.Matrix Biol. 2020; 94: 57-76Google Scholar, 11Izu Y. Adams S.M. Connizzo B.K. Beason D.P. Soslowsky L.J. Koch M. Birk D.E. Collagen XII mediated cellular and extracellular mechanisms regulate establishment of tendon structure and function.Matrix Biol. 2021; 95: 52-67Google Scholar, 12Chiquet M. Birk D.E. Bonnemann C.G. Koch M. Collagen XII: protecting bone and muscle integrity by organizing collagen fibrils.Int J Biochem Cell Biol. 2014; 53: 51-54Google Scholar, 13Izu Y. Sun M. Zwolanek D. Veit G. Williams V. Cha B. Jepsen K.J. Koch M. Birk D.E. Type XII collagen regulates osteoblast polarity and communication during bone formation.J Cell Biol. 2011; 193: 1115-1130Google Scholar Deficiency in collagen XII has been associated with musculoskeletal defects, biomechanical tissue alterations, and impaired wound healing in patients with distal myopathy and a specific type of Ehlers-Danlos syndrome.14Mohassel P. Liewluck T. Hu Y. Ezzo D. Ogata T. Saade D. Neuhaus S. Bolduc V. Zou Y. Donkervoort S. Medne L. Sumner C.J. Dyck P.J.B. Wierenga K.J. Tennekoon G. Finkel R.S. Chen J. Winder T.L. Staff N.P. Foley A.R. Koch M. Bonnemann C.G. Dominant collagen XII mutations cause a distal myopathy.Ann Clin Transl Neurol. 2019; 6: 1980-1988Google Scholar, 15Hicks D. Farsani G.T. Laval S. Collins J. Sarkozy A. Martoni E. Shah A. Zou Y. Koch M. Bonnemann C.G. Roberts M. Lochmuller H. Bushby K. Straub V. Mutations in the collagen XII gene define a new form of extracellular matrix-related myopathy.Hum Mol Genet. 2014; 23: 2353-2363Google Scholar, 16Delbaere S. Dhooge T. Syx D. Petit F. Goemans N. Destree A. Vanakker O. De Rycke R. Symoens S. Malfait F. Novel defects in collagen XII and VI expand the mixed myopathy/Ehlers-Danlos syndrome spectrum and lead to variant-specific alterations in the extracellular matrix.Genet Med. 2020; 22: 112-123Google Scholar In the corneal stroma, collagen XII regulates fibril density, lamellar organization, and tissue biomechanics. This is further supported by a link between decreased collagen XII deposition and keratoconus corneas, a pathologic condition characterized by a weak, ectatic cornea with abnormal distribution and orientation of collagen lamellae.17Meek K.M. Tuft S.J. Huang Y. Gill P.S. Hayes S. Newton R.H. Bron A.J. Changes in collagen orientation and distribution in keratoconus corneas.Invest Ophthalmol Vis Sci. 2005; 46: 1948-1956Google Scholar,18Akhtar S. Bron A.J. Salvi S.M. Hawksworth N.R. Tuft S.J. Meek K.M. Ultrastructural analysis of collagen fibrils and proteoglycans in keratoconus.Acta Ophthalmol. 2008; 86: 764-772Google Scholar The controlled expression and deposition of collagen XII during development emphasizes its important function in regulation and maintenance of tissue matrix architecture and cell behavior11Izu Y. Adams S.M. Connizzo B.K. Beason D.P. Soslowsky L.J. Koch M. Birk D.E. Collagen XII mediated cellular and extracellular mechanisms regulate establishment of tendon structure and function.Matrix Biol. 2021; 95: 52-67Google Scholar,19Gordon M.K. Foley J.W. Lisenmayer T.F. Fitch J.M. Temporal expression of types XII and XIV collagen mRNA and protein during avian corneal development.Dev Dyn. 1996; 206: 49-58Google Scholar,20Gregory K.E. Keene D.R. Tufa S.F. Lunstrum G.P. Morris N.P. Developmental distribution of collagen type XII in cartilage: association with articular cartilage and the growth plate.J Bone Miner Res. 2001; 16: 2005-2016Google Scholar Up-regulation of collagen XII is also detected during wound healing, suggestive of a role in regulating matrix stiffness and organization of newly synthesized matrix.9Massoudi D. Malecaze F. Soler V. Butterworth J. Erraud A. Fournie P. Koch M. Galiacy S.D. NC1 long and NC3 short splice variants of type XII collagen are overexpressed during corneal scarring.Invest Ophthalmol Vis Sci. 2012; 53: 7246-7256Google Scholar,21Tzortzaki E.G. Tischfield J.A. Sahota A. Siafakas N.M. Gordon M.K. Gerecke D.R. Expression of FACIT collagens XII and XIV during bleomycin-induced pulmonary fibrosis in mice.Anat Rec A Discov Mol Cell Evol Biol. 2003; 275: 1073-1080Google Scholar,22Chaerkady R. Shao H. Scott S.G. Pandey A. Jun A.S. Chakravarti S. The keratoconus corneal proteome: loss of epithelial integrity and stromal degeneration.J Proteomics. 2013; 87: 122-131Google Scholar Our Col12a1–/– model, deficient for all collagen isoforms, shows increased fibril density and disorganized stromal lamellae. These changes in matrix organization modify and regulate stromal function (eg, Col12a1–/– stromata have significant structural and mechanical abnormalities, including significant resistance to compression).23Sun M. Zafrullah N. Devaux F. Hemmavanh C. Adams S. Ziebarth N.M. Koch M. Birk D.E. Espana E.M. Collagen XII is a regulator of corneal stroma structure and function.Invest Ophthalmol Vis Sci. 2020; 61: 61Google Scholar The transforming growth factor (TGF)-β system is a major regulator of multiple cell functions such as cell fate, proliferation, movement, polarity, adhesion, cytokine production, terminal differentiation, and cell death.24Massague J. TGF-beta signaling in development and disease.FEBS Lett. 2012; 586: 1833Google Scholar,25David C.J. Massague J. Contextual determinants of TGFbeta action in development, immunity and cancer.Nat Rev Mol Cell Biol. 2018; 19: 419-435Google Scholar Cells secrete TGF-β, a 25-kDa homodimeric protein, but they are not the only regulators of its biological function. TGF-β function is also regulated by its storage and release from the extracellular matrix.26Robertson I.B. Horiguchi M. Zilberberg L. Dabovic B. Hadjiolova K. Rifkin D.B. Latent TGF-beta-binding proteins.Matrix Biol. 2015; 47: 44-53Google Scholar, 27Rifkin D.B. Rifkin W.J. Zilberberg L. LTBPs in biology and medicine: LTBP diseases.Matrix Biol. 2018; 71-72: 90-99Google Scholar, 28Holm T.M. Habashi J.P. Doyle J.J. Bedja D. Chen Y. van Erp C. Lindsay M.E. Kim D. Schoenhoff F. Cohn R.D. Loeys B.L. Thomas C.J. Patnaik S. Marugan J.J. Judge D.P. Dietz H.C. Noncanonical TGFbeta signaling contributes to aortic aneurysm progression in Marfan syndrome mice.Science. 2011; 332: 358-361Google Scholar, 29Robertson I.B. Rifkin D.B. Regulation of the bioavailability of TGF-beta and TGF-beta-related proteins.Cold Spring Harb Perspect Biol. 2016; 8: a021907Google Scholar The large latent complex, a complex of TGF-β, latency-associated protein, and latent TGF-β binding protein (LTBP), plays a critical role in modulating the action of TGF-β1 by controlling its release from the extracellular matrix.26Robertson I.B. Horiguchi M. Zilberberg L. Dabovic B. Hadjiolova K. Rifkin D.B. Latent TGF-beta-binding proteins.Matrix Biol. 2015; 47: 44-53Google Scholar, 27Rifkin D.B. Rifkin W.J. Zilberberg L. LTBPs in biology and medicine: LTBP diseases.Matrix Biol. 2018; 71-72: 90-99Google Scholar, 28Holm T.M. Habashi J.P. Doyle J.J. Bedja D. Chen Y. van Erp C. Lindsay M.E. Kim D. Schoenhoff F. Cohn R.D. Loeys B.L. Thomas C.J. Patnaik S. Marugan J.J. Judge D.P. Dietz H.C. Noncanonical TGFbeta signaling contributes to aortic aneurysm progression in Marfan syndrome mice.Science. 2011; 332: 358-361Google Scholar, 29Robertson I.B. Rifkin D.B. Regulation of the bioavailability of TGF-beta and TGF-beta-related proteins.Cold Spring Harb Perspect Biol. 2016; 8: a021907Google Scholar Tissue fibrosis is characterized by excessive deposition of extracellular matrix proteins, like collagen, which compromises normal tissue structure and function.30Rockey D.C. Bell P.D. Hill J.A. Fibrosis--a common pathway to organ injury and failure.N Engl J Med. 2015; 372: 1138-1149Google Scholar TGF-β is considered to be one of the most potent factors accelerating the progression of tissue fibrosis.31Kitamura H. Cambier S. Somanath S. Barker T. Minagawa S. Markovics J. Goodsell A. Publicover J. Reichardt L. Jablons D. Wolters P. Hill A. Marks J.D. Lou J. Pittet J.F. Gauldie J. Baron J.L. Nishimura S.L. Mouse and human lung fibroblasts regulate dendritic cell trafficking, airway inflammation, and fibrosis through integrin alphavbeta8-mediated activation of TGF-beta.J Clin Invest. 2011; 121: 2863-2875Google Scholar, 32Tandon A. Tovey J.C. Sharma A. Gupta R. Mohan R.R. Role of transforming growth factor beta in corneal function, biology and pathology.Curr Mol Med. 2010; 10: 565-578Google Scholar, 33Hinz B. The extracellular matrix and transforming growth factor-beta1: tale of a strained relationship.Matrix Biol. 2015; 47: 54-65Google Scholar Considering that collagen XII influences the extracellular matrix organization and its potential role during homeostasis and wound healing, the current study was designed to determine whether collagen XII regulates the bioavailability of TGF-β, which is closely associated with hypertrophy, in the corneal stroma. The results demonstrate that collagen XII plays a significant role in regulating TGF-β activity in the corneal stroma and that such regulatory activity has significant effects on stromal structure. The considerable therapeutic potential of this novel regulatory activity of TGF-β signaling in an extracellular context is of major importance and is likely to contribute to corneal wound healing and related fibrotic disorders. Wild-type (WT) and collagen XII–null mice (Col12a1–/–) on C57BL/6 and 129/SvJ mixed backgrounds were used, as previously published.7Hemmavanh C. Koch M. Birk D.E. Espana E.M. Abnormal corneal endothelial maturation in collagen XII and XIV null mice.Invest Ophthalmol Vis Sci. 2013; 54: 3297-3308Google Scholar,23Sun M. Zafrullah N. Devaux F. Hemmavanh C. Adams S. Ziebarth N.M. Koch M. Birk D.E. Espana E.M. Collagen XII is a regulator of corneal stroma structure and function.Invest Ophthalmol Vis Sci. 2020; 61: 61Google Scholar Corneas from mice at age 30 days old, preadult, and ≥60 days old, considered adult, were included in this study. All experiments conformed to the Use of Laboratory Animals and Association for Research in Vision and Ophthalmology statement for the use of animals in ophthalmic and vision research and were approved by the Institutional Animal Care and Use Committee of the University of South Florida College of Medicine. All mice were housed and treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals.34Committee for the Update of the Guide for the Care and Use of Laboratory Animals; National Research CouncilGuide for the Care and Use of Laboratory Animals: Eighth Edition. National Academies Press, Washington, DC2011Google Scholar Fresh eyes were harvested from C57BL/6 mice at preadult and adult age. They were then embedded in OCT compound and frozen with isopentane (Sigma Aldrich, St. Louis, MO) on dry ice. Corneal sections (5 to 7 μm thick) were blocked using 10% donkey serum (Sigma Aldrich). DAPI Fluoromount-G clear mounting solution (SouthernBiotech, Birmingham, AL) with DAPI was used as a nuclear marker. The number of nuclei in the central cornea were counted in a ×20 magnification field. At least three different animals per age and condition were used. Whole eyes from euthanized mice were enucleated, and measurements were immediately obtained. Each enucleated eye was placed in a custom-made holder and placed in the Spectral Dominium Cirrus HDT Optical Coherence Tomography (Zeiss, San Francisco, CA) device for corneal thickness measurements.7Hemmavanh C. Koch M. Birk D.E. Espana E.M. Abnormal corneal endothelial maturation in collagen XII and XIV null mice.Invest Ophthalmol Vis Sci. 2013; 54: 3297-3308Google Scholar Five measurements were obtained on the vertical plane, and five measurements were obtained on the horizontal plane of the central cornea. All experiments were performed at least three times in adult corneas, where total endothelial maturation and function was achieved. The measurement of total collagen deposited in the mouse cornea was performed, as previously described.35Segev F. Heon E. Cole W.G. Wenstrup R.J. Young F. Slomovic A.R. Rootman D.S. Whitaker-Menezes D. Chervoneva I. Birk D.E. Structural abnormalities of the cornea and lid resulting from collagen V mutations.Invest Ophthalmol Vis Sci. 2006; 47: 565-573Google Scholar Basically, the total collagen was quantified indirectly by a colorimetric hydroxyproline assay that generates a chromophore from hydroxyproline via reaction with p-dimethylaminobenzaldehyde (alias Ehrlich's reagent). Corneas were dissected from WT and Col12a1–/– adult mice. Samples were hydrolyzed in 6 N HCl at 100°C for 24 hours, and vacuum dried through NaOH trap, then resuspended with 0.5 mL 1 mmol/L HCl. Hydroxyproline amino acids in 0.2-mL diluted samples were converted to pyrolle-2-carboxylate by oxidation via addition of 0.1 mL of 0.06 mol/L chloramine-T in a buffer containing 70% v/v H2O, 30% v/v 2-propanol, and acetate-citrate buffer (pH 6.0) by incubation at room temperature for 20 minutes. Finally, 1.3 mL of 6.25% w/v p-dimethylaminobenzaldehyde in 2-propanol plus perchlorate acid (alias Ehrlich's solution) was added to each sample, mixed well, incubated at 55°C for 20 minutes, and cooled, and then absorbance was determined at 550 nm. The hydroxyproline concentration was determined from a standard curve (stock solution of hydroxyproline: 1 mg/mL in 1 mmol/L HCl). The mean collagen content from each cornea was calculated by using a conversion ratio of 0.125:1.0 to convert micrograms of hydroxyproline to total collagen. Freshly enucleated eyes were immediately placed on Optisol media on a custom-made glass slide and imaged without any tissue manipulation or further tissue dissection within 5 minutes of enucleation. Adult corneas, age postnatal day 60, were dissected from the globe and placed as a flat mount for en face imaging. Corneal cross-sections were imaged using an Olympus MPE-RS microscope using a 25× (0.95 numerical aperture) water-immersion objective (Olympus Corp., Tokyo, Japan). Two-photon second harmonic generation (SHG) signals were generated using a mode-locked titanium/sapphire laser at 960 nm. The SHG forward-scattered signals passing through the corneal sections were collected using a 0.8 numerical aperture condenser lens with a narrow band-pass filter (465 to 485 nm). Backward-scattered SHG signals were detected with a band-pass filter (460 to 500 nm). All samples were scanned using a 2-μm z-axis step size from the back to the front of the section. All of the experimental settings and conditions were kept constant throughout the experiment. After euthanasia, the eyes of adult WT mice were copiously washed with betadine ophthalmic solution, and then incubated in Dulbecco’s modified Eagle’s medium (DMEM) containing 15 mg/mL dispase II (Roche Applied Science, Penzberg, Germany) at 4°C for 18 hours. The entire corneal epithelium sheet loosened by this treatment was removed by vigorous shaking. Under a dissecting microscope, the corneal stroma was separated from the sclera at the corneoscleral limbus by pressing down the limbus with a 27-gauge needle. Isolated corneal stromata were incubated overnight at 37°C in DMEM containing 1.25 mg/mL collagenase A (Roche Applied Science) and 25 μg/mL gentamicin. A keratocyte-containing cell suspension was then seeded on T25 flasks (Thermo Fisher Scientific, Waltham, MA) in DMEM containing ITS (5 μg/mL insulin, 5 μg/mL transferrin, and 5 ng/mL sodium selenite), and 25 μg/mL gentamicin supplemented with 5% fetal bovine serum (FBS). The suspension of keratocytes prepared from 12 mouse corneal buttons was seeded into each flask. Whole corneas were dissected from preadult WT mice and Col12a1–/– mice, cut into small pieces, and total RNA was extracted using QIAzol Lysis Reagent (Qiagen, Hilden, Germany) and the Qiagen RNeasy MinElute Cleanup Kit (Qiagen, Venio, the Netherlands). Reverse transcription and quantitative real-time PCR analyses were performed, as previously described.23Sun M. Zafrullah N. Devaux F. Hemmavanh C. Adams S. Ziebarth N.M. Koch M. Birk D.E. Espana E.M. Collagen XII is a regulator of corneal stroma structure and function.Invest Ophthalmol Vis Sci. 2020; 61: 61Google Scholar The primer sequences used are listed in Table 1. Each sample was run in a duplicate PCR, and statistical analysis was performed on six or seven corneas from different mice.Table 1Primers Used in This ArticleGeneForward primerReverse primerSerpine15′-GTCTTTCCGACCAAGAGCAG-3′5′-GACAAAGGCTGTGGAGGAAG-3′Col1a15′-TTCTCCTGGCAAAGACGGACTCAA-3′5′-AGGAAGCTGAAGTCATAACCGCCA-3′Col5a15′-AAGCGTGGGAAACTGCTCTCCTAT-3′5′-AGCAGTTGTAGGTGACGTTCTGGT-3′Col12a15′-CCCTACAACAGATGGGCCTAC-3′5′-TCTTCTCCCCTGGCTTTGTA-3′Lox5′-CAGAGGAGAGTGGCTGAAGG-3′5′-CTCAATCCCTGTGTGTGTGC-3′Actb5′-AGATGACCCAGATCATGTTTGAGA-3′5′-CACAGCCTGGATGGCTACGT-3′ Open table in a new tab Constitutive lentiviral miR-E expression vector36Fellmann C. Hoffmann T. Sridhar V. Hopfgartner B. Muhar M. Roth M. Lai D.Y. Barbosa I.A. Kwon J.S. Guan Y. Sinha N. Zuber J. An optimized microRNA backbone for effective single-copy RNAi.Cell Rep. 2013; 5: 1704-1713Google Scholar featuring puromycin drug selection and green fluorescent protein fluorescent marker was a gift from Dr. Florian Karreth at Moffitt Cancer Center and Research Institute (Tampa, FL). De novo 97-mer oligo was designed by using SplashRNA shRNA prediction tool (http://splashrna.mskcc.org, last accessed March 29, 2021).37Pelossof R. Fairchild L. Huang C.H. Widmer C. Sreedharan V.T. Sinha N. Lai D.Y. Guan Y. Premsrirut P.K. Tschaharganeh D.F. Hoffmann T. Thapar V. Xiang Q. Garippa R.J. Ratsch G. Zuber J. Lowe S.W. Leslie C.S. Fellmann C. Prediction of potent shRNAs with a sequential classification algorithm.Nat Biotechnol. 2017; 35: 350-353Google Scholar Three oligonucleotides (1833, 5′-TGCTGTTGACAGTGAGCGAAGAGTTGAAGATATAATCAAATAGT-GAAGCCACAGATGTATTTGATTATATCTTCAACTCT-GTGCCTACTGCCTCGGA-3′; 5121, 5′-TGCTGTTGACAGTGAGCGAAAGTACATTGTTAGATACAAATAGT-GAAGCCACAGATGTATTTGTATCTAACAATGTACT-TGTGCCTACTGCCTCGGA-3′; and 902, 5′-TGCTGTTG-ACAGTGAGCGACAGGACTGAATTTAACTTAAATA-GTGAAGCCACAGATGTATTTAAGTTAAATTCAGT-CCTGGTGCCTACTGCCTCGGA-3′; Sigma, St. Louis, MO) coding for Col12a1 shRNAs were PCR amplified using the primers miRE-Xho-fw (5′-TGAACTCGAGAAGGTATATTGCTGTTGACAGTGAGCG-3′) and miRE-EcoOligo-rev (5′-TCTCGAATTCTAGCCCCTTGAAGTCCGAGGCAGTAGGC-3′), 0.05 ng oligonucleotide template, and the PfuUltra HF kit (Agilent Technologies, Santa Clara, CA), and cloned into XhoI/EcoRI sites of miR-E recipient vectors. An oligonucleotide-targeting Renilla luciferase (Ren.713) was used as a negative control. Plasmid DNA was amplified and purified using a HiSpeed Plasmid Midi kit (Qiagen, Hilden, Germany) and then transfected along with packaging plasmid using JetPrime (Polyplus Transfection, New York, NY) into HEK293T cells to generate lentiviruses. The cells were refed with 1.5 mL DMEM supplemented with 10% FBS and 1× antibiotic-antimycotic (Thermo Fisher, Waltham, MA) 24 and 48 hours after transfection. The culture supernatant was harvested 72 hours after transfection. The supernatant-containing lentivirus was filtered and used to infect the mouse corneal fibroblast cells. The corneal fibroblast cells were cultured with DMEM supplemented with 10% FBS and 1× antibiotic-antimycotic, as well as 2 μg/mL puromycin, to select target cells. After selection for 7 days, cells were analyzed for real-time PCR (see primer sequence in Table 1) and protein immunoblotting analysis, using rabbit anti–type XII collagen antibody (1:1000 dilution; KR33) and mouse anti–β-actin (1:1000 dilution; Millipore, Burlington, MA). Transformed mink lung cells, transfected with luciferase cDNA driven by plasminogen activator inhibitor (PAI-1) promoter, were a generous gift of Dr. Daniel Rifkin (New York University, New York, NY).38Abe M. Harpel J.G. Metz C.N. Nunes I. Loskutoff D.J. Rifkin D.B. An assay for transforming growth factor-beta using cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct.Anal Biochem. 1994; 216: 276-284Google Scholar Cells were cultured in DMEM supplemented with 10% FBS (Thermo Fisher). The amount of latent TGF-β in corneas isolated from Col12a1–/– and WT corneas was calculated on the basis of a comparison to a standard curve generated using different concentrations of human recombinant TGF-β1 (Sigma). For analysis and quantification of latent TGF-β, corneal stromata were minced into 10 to 12 little pieces with a blade and heated at 80°C for 10 minutes to release latent TGF-β. Equal amounts of protein were added to transformed mink lung cells for incubation for 16 hours under serum-free conditions. For analysis and quantification of active TGF-β, a co-culture system was performed, transformed mink lung cells were seeded and allowed to attach for 4 hours, and then corneal fibroblasts were seeded in serum-free DMEM and incubated for 16 hours. TGF-β signaling was inhibited by the addition of the TGF-β type I receptor/activin receptor-like kinase 5 (ALK5) inhibitor, SB431542 (Tocris Bioscience, Minneapolis, MN) to 10 μmol/L final concentration. A neutralization antibody against all three TGF-β isoforms (clone 1D11; R&D System, Minneapolis, MN) was also used. Luciferase assay was performed by using Promega's Luciferase Assay System (Promega, Madison, WI), and luminance was measured with Synergy HT plate reader (BIO-TEK, Winooski, VT). Total TGF-β1 antigen in mouse fibroblast culture medium was evaluated using a commercial kit, Quantikine Mouse TGF-β1 Immunoassay (R&D Systems; catalog number MB100B), according to the manufacturer's instructions. This enzyme-linked immunosorbent assay (ELISA) is used to measure active plus acid-activatable latent TGF-β1 in the cell culture supernatant. To activate the latent TGF-β1 form, each sample was acidified by 1 N HCL for 10 minutes, which was followed by neutralization by 1.2 N NaOH/0.5 mol/L HEPES. Because samples have been diluted in the activation step, the concentration read from the standard curve was multiplied by the dilution factor 1.4. All standards and samples were tested in duplicate, and the mean values were used for calculation. Same number of WT and Col12a1−/− keratocytes cells were seeded in a 6-well plate, with 3 wells for each genotype. Cells were cultured in DMEM supplemented with 1% FBS and ITS (1.0 mg/mL recombinant human insulin, 0.55 mg/mL human transferrin, and 0.5 μg/mL sodium selenite at the 100× concentration; Sigma) serum-free medium and 50 μg/mL ascorbic acid for 5 days. One day before the ELISA assay, cells were maintained in serum-free DMEM medium 24 hours before collecting medium. Data are presented as mean ± SD. t-Test was used to draw statistical inferences when comparing the mean of continuous dependent variables. P < 0.05 was considered statistically significant. Graph Pad Prism software version 9.1.2 (San Diego, CA) was used for statistical calculations. Histologic observation of stromal morphology in Col12a1−/− and WT mice at different ages suggested that collagen XII influences the number of keratocytes, as well as the thickness of stromal tissue (Figure 1, A and B). Cell density and central corneal thickness were analyzed. Keratocyte density was evaluated by counting keratocytes in corneal cross-sections at different ages in the WT and Col12a1−/− stromata. A hyperplastic number of keratocytes was found in the Col12a1−/− stromata in the preadult age. Keratocytes per ×20 field were 82.4 ± 11.1 in Col12a1−/− versus 59 ± 8.5 in WT stromata. A statistically significant difference was found between Col12a1−/− and WT (unpaired t" @default.
• W3213680757 created "2021-11-22" @default.
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• W3213680757 date "2022-02-01" @default.
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• W3213680757 title "Collagen XII Regulates Corneal Stromal Structure by Modulating Transforming Growth Factor-β Activity" @default.
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• W3213680757 doi "https://doi.org/10.1016/j.ajpath.2021.10.014" @default.
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