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Zhou, X., Li, J., Backman, L. J. & Danielson, P. (2022). Keratocyte Differentiation Is Regulated by NF-κB and TGFβ Signaling Crosstalk. International Journal of Molecular Sciences, 23(19), Article ID 11073.
Åpne denne publikasjonen i ny fane eller vindu >>Keratocyte Differentiation Is Regulated by NF-κB and TGFβ Signaling Crosstalk
2022 (engelsk)Inngår i: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 23, nr 19, artikkel-id 11073Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Interleukin-1 (IL-1) and transforming growth factor-beta (TGFβ) are important cytokines involved in corneal wound healing. Here, we studied the effect of these cytokines on corneal stromal cell (keratocyte) differentiation. IL-1β treatment resulted in reduced keratocyte phenotype, as evident by morphological changes and decreased expression of keratocyte markers, including keratocan, lumican, ALDH3A1, and CD34. TGFβ1 treatment induced keratocyte differentiation towards the myofibroblast phenotype. This was inhibited by simultaneous treatment with IL-1β, as seen by inhibition of α-SMA expression, morphological changes, and reduced contractibility. We found that the mechanism of crosstalk between IL-1β and TGFβ1 occurred via regulation of the NF-κB signaling pathway, since the IL-1β induced inhibition of TGFβ1 stimulated keratocyte-myofibroblast differentiation was abolished by a specific NF-κB inhibitor, TPCA-1. We further found that Smad7 participated in the downstream signaling. Smad7 expression level was negatively regulated by IL-1β and positively regulated by TGFβ1. TPCA-1 treatment led to an overall upregulation of Smad7 at mRNA and protein level, suggesting that NF-κB signaling downregulates Smad7 expression levels in keratocytes. All in all, we propose that regulation of cell differentiation from keratocyte to fibroblast, and eventually myofibroblast, is closely related to the opposing effects of IL-1β and TGFβ1, and that the mechanism of this is governed by the crosstalk of NF-κB signaling.

sted, utgiver, år, opplag, sider
MDPI, 2022
Emneord
corneal wound healing, IL-1, keratocyte, NF-κB, TGFβ
HSV kategori
Identifikatorer
urn:nbn:se:umu:diva-200563 (URN)10.3390/ijms231911073 (DOI)000867737100001 ()36232373 (PubMedID)2-s2.0-85139931390 (Scopus ID)
Forskningsfinansiär
Swedish Research Council, 2017-01138Stiftelsen Kronprinsessan Margaretas arbetsnämnd för synskadade, 2013/10Umeå UniversityRegion Västerbotten, RV-930288
Tilgjengelig fra: 2022-12-13 Laget: 2022-12-13 Sist oppdatert: 2023-10-18bibliografisk kontrollert
Prittinen, J., Zhou, X., Bano, F., Backman, L. J. & Danielson, P. (2022). Microstructured collagen films for 3D corneal stroma modelling. Connective Tissue Research, 63(5), 443-452
Åpne denne publikasjonen i ny fane eller vindu >>Microstructured collagen films for 3D corneal stroma modelling
Vise andre…
2022 (engelsk)Inngår i: Connective Tissue Research, ISSN 0300-8207, E-ISSN 1607-8438, Vol. 63, nr 5, s. 443-452Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Purpose/aim: Corneal injury is a major cause of impaired vision around the globe. The fine structure of the corneal stroma plays a pivotal role in the phenotype and behavior of the embedded cells during homeostasis and healing after trauma or infection. In order to study healing processes in the cornea, it is important to create culture systems that functionally mimic the natural environment.

Materials and methods: Collagen solution was vitrified on top of a grated film to achieve thin collagen films with parallel microgrooves. Keratocytes (corneal stromal cells) were cultured on the films either as a single layer or as stacked layers of films and cells. SEM and F-actin staining were used to analyze the pattern transference onto the collagen and the cell orientation on the films. Cell viability was analyzed with MTS and live/dead staining. Keratocytes, fibroblasts, and myofibroblasts were cultured to study the pattern’s effect on phenotype.

Results: A microstructured collagen film-based culture system that guides keratocytes (stromal cells) to their native, layerwise perpendicular orientation in 3D and that can support fibroblasts and myofibroblasts was created. The films are thin and transparent enough to observe cells at least three layers deep. The cells maintain viability in 2D and 3D cultures and the films can support fibroblast and myofibroblast phenotypes.

Conclusions: The films provide an easily reproducible stroma model that maintains high cell viability and improves the preservation of the keratocyte phenotype in keratocytes that are differentiated to fibroblasts.

sted, utgiver, år, opplag, sider
Taylor & Francis Group, 2022
Emneord
collagen, cornea, keratocyte, stroma, Vitrigel
HSV kategori
Identifikatorer
urn:nbn:se:umu:diva-190877 (URN)10.1080/03008207.2021.2007901 (DOI)000729669400001 ()34894951 (PubMedID)2-s2.0-85121425675 (Scopus ID)
Forskningsfinansiär
Stiftelsen Kronprinsessan Margaretas arbetsnämnd för synskadade, 2013/10Swedish Society of Medicine, 504541Swedish Research Council, 2017-01138Region Västerbotten, 549761
Tilgjengelig fra: 2021-12-29 Laget: 2021-12-29 Sist oppdatert: 2024-07-02bibliografisk kontrollert
Zhou, X., Backman, L. J. & Danielson, P. (2021). Activation of NF-κB signaling via cytosolic mitochondrial RNA sensing in kerotocytes with mitochondrial DNA common deletion. Scientific Reports, 11(1), Article ID 7360.
Åpne denne publikasjonen i ny fane eller vindu >>Activation of NF-κB signaling via cytosolic mitochondrial RNA sensing in kerotocytes with mitochondrial DNA common deletion
2021 (engelsk)Inngår i: Scientific Reports, E-ISSN 2045-2322, Vol. 11, nr 1, artikkel-id 7360Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Scar formation as a result of corneal wound healing is a leading cause of blindness. It is a challenge to understand why scar formation is more likely to occur in the central part of the cornea as compared to the peripheral part. The purpose of this study was to unravel the underlying mechanisms. We applied RNA-seq to uncover the differences of expression profile in keratocytes in the central/peripheral part of the cornea. The relative quantity of mitochondrial RNA was measured by multiplex qPCR. The characterization of mitochondrial RNA in the cytoplasm was confirmed by immunofluoresence microscope and biochemical approach. Gene expression was analyzed by western blot and RT qPCR. We demonstrate that the occurrence of mitochondrial DNA common deletion is greater in keratocytes from the central cornea as compared to those of the peripheral part. The keratocytes with CD have elevated oxidative stress levels, which leads to the leakage of mitochondrial double-stranded RNA into the cytoplasm. The cytoplasmic mitochondrial double-stranded RNA is sensed by MDA5, which induces NF-κB activation. The NF-κB activation thereafter induces fibrosis-like extracellular matrix expressions and IL-8 mRNA transcription. These results provide a novel explanation of the different clinical outcome in different regions of the cornea during wound healing.

sted, utgiver, år, opplag, sider
Nature Publishing Group, 2021
HSV kategori
Identifikatorer
urn:nbn:se:umu:diva-182367 (URN)10.1038/s41598-021-86522-6 (DOI)000636796200002 ()33795727 (PubMedID)2-s2.0-85103674533 (Scopus ID)
Tilgjengelig fra: 2021-04-26 Laget: 2021-04-26 Sist oppdatert: 2023-10-18bibliografisk kontrollert
Sloniecka, M. & Danielson, P. (2020). Acetylcholine decreases formation of myofibroblasts and excessive extracellular matrix production in an in vitro human corneal fibrosis model. Journal of Cellular and Molecular Medicine (Print), 24(8), 4850-4862
Åpne denne publikasjonen i ny fane eller vindu >>Acetylcholine decreases formation of myofibroblasts and excessive extracellular matrix production in an in vitro human corneal fibrosis model
2020 (engelsk)Inngår i: Journal of Cellular and Molecular Medicine (Print), ISSN 1582-1838, E-ISSN 1582-4934, Vol. 24, nr 8, s. 4850-4862Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Acetylcholine (ACh) has been reported to play various physiological roles, including wound healing in the cornea. Here, we study the role of ACh in the transition of corneal fibroblasts into myofibroblasts, and in consequence its role in the onset of fibrosis, in an in vitro human corneal fibrosis model. Primary human keratocytes were obtained from healthy corneas. Vitamin C (VitC) and transforming growth factor-β1 (TGF-β1) were used to induce fibrosis in corneal fibroblasts. qRT-PCR and ELISA analyses showed that gene expression and production of collagen I, collagen III, collagen V, lumican, fibronectin (FN) and alpha-smooth muscle actin (α-SMA) were reduced by ACh in quiescent keratocytes. ACh treatment furthermore decreased gene expression and production of collagen I, collagen III, collagen V, lumican, FN and α-SMA during the transition of corneal fibroblasts into myofibroblasts, after induction of fibrotic process. ACh inhibited corneal fibroblasts from developing contractile activity during the process of fibrosis, as assessed with collagen gel contraction assay. Moreover, the effect of ACh was dependent on activation of muscarinic ACh receptors. These results show that ACh has an anti-fibrotic effect in an in vitro human corneal fibrosis model, as it negatively affects the transition of corneal fibroblasts into myofibroblasts. Therefore, ACh might play a role in the onset of fibrosis in the corneal stroma.

sted, utgiver, år, opplag, sider
John Wiley & Sons, 2020
Emneord
collagens, cornea, fibrotic markers, keratocytes, scarring
HSV kategori
Identifikatorer
urn:nbn:se:umu:diva-170515 (URN)10.1111/jcmm.15168 (DOI)000527848000043 ()32176460 (PubMedID)2-s2.0-85081750271 (Scopus ID)
Tilgjengelig fra: 2020-05-07 Laget: 2020-05-07 Sist oppdatert: 2023-03-24bibliografisk kontrollert
Chen, J., Backman, L. J., Zhang, W., Ling, C. & Danielson, P. (2020). Regulation of Keratocyte Phenotype and Cell Behavior by Substrate Stiffness. ACS Biomaterials Science & Engineering, 6(9), 5162-5171
Åpne denne publikasjonen i ny fane eller vindu >>Regulation of Keratocyte Phenotype and Cell Behavior by Substrate Stiffness
Vise andre…
2020 (engelsk)Inngår i: ACS Biomaterials Science & Engineering, E-ISSN 2373-9878, Vol. 6, nr 9, s. 5162-5171Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Corneal tissue engineering is an alternative way to solve the problem of lack of corneal donor tissue in corneal transplantation. Keratocytes with a normal phenotype and function in tissue-engineered cornea would be critical for corneal regeneration. Although the role of extracellular/substrate material stiffness is well-known for the regulation of the cell phenotype and cell behavior in many different cell types, its effects in keratocyte culture have not yet been thoroughly studied. This project studied the effect of substrate stiffness on the keratocyte phenotype marker expression and typical cell behavior (cell adhesion, proliferation, and migration), and the possible mechanisms involved. Human primary keratocytes were cultured on tissue culture plastic (TCP, similar to 10(6) kPa) or on plates with the stiffness equivalent of physiological human corneal stroma (25 kPa) or vitreous body (1 kPa). The expression of keratocyte phenotype markers, cell adhesion, proliferation, and migration were compared. The results showed that the stiffness of the substrate material regulates the phenotype marker expression and cell behavior of cultured keratocytes. Physiological corneal stiffness (25 kPa) superiorly preserved the cell phenotype when compared to the TCP and 1 kPa group. Keratocytes had a larger cell area when cultured on 25 kPa plates as compared to on TCP. Treatment of cells with NSC 23766 (Rac1 inhibitor) mimicked the response in the cell phenotype and behavior seen in the transition from soft materials to stiff materials, including the cytoskeletal structure, expression of keratocyte phenotype markers, and cell behavior. In conclusion, this study shows that substrate stiffness regulates the cell phenotype marker expression and cell behavior of keratocytes by Rac1-mediated cytoskeletal reorganization. This knowledge contributes to the development of corneal tissue engineering.

sted, utgiver, år, opplag, sider
American Chemical Society (ACS), 2020
Emneord
keratocytes, stiffness, phenotype, cell behavior, cytoskeletal reorganization, Rac1
HSV kategori
Identifikatorer
urn:nbn:se:umu:diva-176078 (URN)10.1021/acsbiomaterials.0c00510 (DOI)000572822300037 ()2-s2.0-85092544653 (Scopus ID)
Tilgjengelig fra: 2020-10-23 Laget: 2020-10-23 Sist oppdatert: 2023-03-23bibliografisk kontrollert
Zhang, W., Chen, J., Qu, M., Backman, L. J., Zhang, A., Liu, H., . . . Danielson, P. (2020). Sustained Release of TPCA-1 from Silk Fibroin Hydrogels Preserves Keratocyte Phenotype and Promotes Corneal Regeneration by Inhibiting Interleukin-1β Signaling. Advanced Healthcare Materials, 9(17), Article ID 2000591.
Åpne denne publikasjonen i ny fane eller vindu >>Sustained Release of TPCA-1 from Silk Fibroin Hydrogels Preserves Keratocyte Phenotype and Promotes Corneal Regeneration by Inhibiting Interleukin-1β Signaling
Vise andre…
2020 (engelsk)Inngår i: Advanced Healthcare Materials, ISSN 2192-2640, E-ISSN 2192-2659, Vol. 9, nr 17, artikkel-id 2000591Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Corneal injury due to ocular trauma or infection is one of the most challenging vision impairing pathologies that exists. Many studies focus on the pro-inflammatory and pro-angiogenic effects of interleukin-1 beta(IL-1 beta) on corneal wound healing. However, the effect of IL-1 beta on keratocyte phenotype and corneal repair, as well as the underlying mechanisms, is not clear. This study reports, for the first time, that IL-1 beta induces phenotype changes of keratocytes in vitro, by significantly down-regulating the gene and protein expression levels of keratocyte markers (Keratocan, Lumican, Aldh3a1 and CD34). Furthermore, it is found that the NF-kappa B pathway is involved in the IL-1 beta-induced changes of keratocyte phenotype, and that the selective IKK beta inhibitor TPCA-1, which inhibits NF-kappa B, can preserve keratocyte phenotype under IL-1 beta simulated pathological conditions in vitro. By using a murine model of corneal injury, it is shown that sustained release of TPCA-1 from degradable silk fibroin hydrogels accelerates corneal wound healing, improves corneal transparency, enhances the expression of keratocyte markers, and supports the regeneration of well-organized epithelium and stroma. These findings provide insights not only into the pathophysiological mechanisms of corneal wound healing, but also into the potential development of new treatments for patients with corneal injuries.

sted, utgiver, år, opplag, sider
Wiley-VCH Verlagsgesellschaft, 2020
Emneord
corneal regeneration, interleukin-1 beta, keratocyte, NF-kappa B signaling, silk fibroin
HSV kategori
Identifikatorer
urn:nbn:se:umu:diva-174033 (URN)10.1002/adhm.202000591 (DOI)000554483600001 ()32743953 (PubMedID)2-s2.0-85088834694 (Scopus ID)
Tilgjengelig fra: 2020-08-14 Laget: 2020-08-14 Sist oppdatert: 2023-03-24bibliografisk kontrollert
Wang, X., Qu, M., Li, J., Danielson, P., Yang, L. & Zhou, Q. (2019). Induction of Fibroblast Senescence During Mouse Corneal Wound Healing. Investigative Ophthalmology and Visual Science, 60(10), 3669-3679
Åpne denne publikasjonen i ny fane eller vindu >>Induction of Fibroblast Senescence During Mouse Corneal Wound Healing
Vise andre…
2019 (engelsk)Inngår i: Investigative Ophthalmology and Visual Science, ISSN 0146-0404, E-ISSN 1552-5783, Vol. 60, nr 10, s. 3669-3679Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

PURPOSE. To investigate the presence and role of fibroblast senescence in the dynamic process of corneal wound healing involving stromal cell apoptosis, proliferation, and differentiation.

METHODS. An in vivo corneal wound healing model was performed using epithelial debridement in C57BL/6 mice. The corneas were stained using TUNEL, Ki67, and alpha-smooth muscle actin (alpha-SMA) as markers of apoptosis, proliferation, and myofibroblastic differentiation, respectively. Cellular senescence was confirmed by senescence-associated beta-galactosidase (SA-beta-gal) staining and P16(Ink4a) expression. Mitogenic response and gene expression were compared among normal fibroblasts, H2O2-induced senescent fibroblasts, and TGF-beta-induced myofibroblasts in vitro. The senescence was further detected in mouse models of corneal scarring, alkali burn, and penetrating keratoplasty (PKP).

RESULTS. The apoptosis and proliferation of corneal stromal cells were found to peak at 4 and 24 hours after epithelial debridement. Positive staining of SA-beta-gal was observed clearly in the anterior stromal cells at 3 to 5 days. The senescent cells displayed P16(Ink4a) thorn vimentin+ alpha-SMA+, representing the major origin of activated corneal resident fibroblasts. Compared with normal fibroblasts and TGF-beta-induced myofibroblasts, H2O2-induced senescent fibroblasts showed a nonfibrogenic phenotype, including a reduced response to growth factor basic fibroblast growth factor (bFGF) or platelet-derived growth factor-BB (PDGF-BB), increased matrix metalloproteinase (MMP) 1/3/13 expression, and decreased fibronectin and collagen I expression. Moreover, cellular senescence was commonly found in the mouse corneal scarring, alkali burn, and PKP models.

CONCLUSIONS. Corneal epithelial debridement induced the senescence of corneal fibroblasts after apoptosis and proliferation. The senescent cells displayed a nonfibrogenic phenotype and may be involved in the self-limitation of corneal fibrosis.

sted, utgiver, år, opplag, sider
ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2019
Emneord
corneal wound healing, senescence, apoptosis, proliferation, nonfibrogenic, SMOULIERE A, 1995, AMERICAN JOURNAL OF PATHOLOGY, V146, P56 gunawela Romesh I., 2011, JOURNAL OF REFRACTIVE SURGERY, V27, P111 ng Lingling, 2019, STEM CELLS TRANSLATIONAL MEDICINE, V8, P46 tthaei Mario, 2014, EXPERIMENTAL EYE RESEARCH, V129, P13 ao Xiaowen, 2016, BMC OPHTHALMOLOGY, V16, lson Steven E., 2007, EXPERIMENTAL EYE RESEARCH, V85, P305 mura T, 2006, INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE, V47, P1387 e Wen, 2007, NATURE, V445, P656 ilds Bennett G., 2016, SCIENCE, V354, P472 llego-Munoz Patricia, 2017, CYTOKINE, V96, P94 heredge LaTia, 2009, INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE, V50, P3128 vak JM, 2002, PROGRESS IN RETINAL AND EYE RESEARCH, V21, P1 e-Won K, 2015, Nature, V4, P547 ar Marjolein P., 2017, CELL, V169, P132 mpisi Judith, 2007, NATURE REVIEWS MOLECULAR CELL BIOLOGY, V8, P729 n Deursen Jan M., 2014, NATURE, V509, P439 yer Kathleen, 2016, JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY, V67, P2018
HSV kategori
Identifikatorer
urn:nbn:se:umu:diva-164543 (URN)10.1167/iovs.19-26983 (DOI)000483885400008 ()31469894 (PubMedID)2-s2.0-85071748495 (Scopus ID)
Tilgjengelig fra: 2019-11-14 Laget: 2019-11-14 Sist oppdatert: 2023-03-23bibliografisk kontrollert
Sloniecka, M. & Danielson, P. (2019). Substance P induces fibrotic changes through activation of the RhoA/ROCK pathway in an in vitro human corneal fibrosis model. Journal of Molecular Medicine, 97(10), 1477-1489
Åpne denne publikasjonen i ny fane eller vindu >>Substance P induces fibrotic changes through activation of the RhoA/ROCK pathway in an in vitro human corneal fibrosis model
2019 (engelsk)Inngår i: Journal of Molecular Medicine, ISSN 0946-2716, E-ISSN 1432-1440, Vol. 97, nr 10, s. 1477-1489Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Fibrosis is characterized by hardening, overgrowth, and development of scars in various tissues as a result of faulty reparative processes, diseases, or chronic inflammation. During the fibrotic process in the corneal stroma of the eye, the resident cells called keratocytes differentiate into myofibroblasts, specialized contractile fibroblastic cells that produce excessive amounts of disorganized extracellular matrix (ECM) and pro-fibrotic components such as alpha-smooth muscle actin (alpha-SMA) and fibronectin. This study aimed to elucidate the role of substance P (SP), a neuropeptide that has been shown to be involved in corneal wound healing, in ECM production and fibrotic markers expression in quiescent human keratocytes, and during the onset of fibrosis in corneal fibroblasts, in an in vitro human corneal fibrosis model. We report that SP induces keratocyte contraction and upregulates gene expression of collagens I, III, and V, and fibrotic markers: alpha-SMA and fibronectin, in keratocytes. Using our in vitro human corneal fibrosis model, we show that SP enhances gene expression and secretion of collagens I, III, and V, and lumican. Moreover, SP upregulates gene expression and secretion of alpha-SMA and fibronectin, and increases contractility of corneal fibroblasts during the onset of fibrosis. Activation of the preferred SP receptor, the neurokinin-1 receptor (NK-1R), is necessary for the SP-induced pro-fibrotic changes. In addition, SP induces the pro-fibrotic changes through activation of the RhoA/ROCK pathway. Taken together, we show that SP has a pro-fibrotic effect in both quiescent human keratocytes and during the onset of fibrosis in an in vitro human corneal fibrosis model.

sted, utgiver, år, opplag, sider
Springer Berlin/Heidelberg, 2019
Emneord
Cornea, Keratocytes, Scarring, Collagens, Fibrotic markers
HSV kategori
Identifikatorer
urn:nbn:se:umu:diva-164380 (URN)10.1007/s00109-019-01827-4 (DOI)000486177200009 ()31399750 (PubMedID)2-s2.0-85070259214 (Scopus ID)
Tilgjengelig fra: 2019-11-12 Laget: 2019-11-12 Sist oppdatert: 2023-03-24bibliografisk kontrollert
Sloniecka, M. & Danielson, P. (2018). Acetylcholine decreases formation of myofibroblasts and excessive extracellular matrix components production in an in vitro human corneal fibrosis model. Paper presented at Annual Meeting of the Association-for-Research-in-Vision-and-Ophthalmology (ARVO), APR 29-MAY 03, 2018, Honolulu, HI. Investigative Ophthalmology and Visual Science, 59(9)
Åpne denne publikasjonen i ny fane eller vindu >>Acetylcholine decreases formation of myofibroblasts and excessive extracellular matrix components production in an in vitro human corneal fibrosis model
2018 (engelsk)Inngår i: Investigative Ophthalmology and Visual Science, ISSN 0146-0404, E-ISSN 1552-5783, Vol. 59, nr 9Artikkel i tidsskrift, Meeting abstract (Annet vitenskapelig) Published
Abstract [en]

Purpose : Acetylcholine (ACh) is a neurotransmitter present in corneal stroma and produced by keratocytes. It has been shown to play a role in processes important for wound healing. Based on literature and our previous studies, we hypothesize that ACh regulates expression of extracellular matrix (ECM) components that are overexpressed during fibrosis, such as collagens, proteoglycans, fibronectin and metalloproteinases, in a protective manner during corneal fibrosis, i.e. decreasing their expression.

Methods : Primary keratocytes were isolated from healthy human corneas obtained from the local cornea bank and grown in presence of 10% fetal bovine serum in order to obtain corneal fibroblasts. A corneal fibrosis in vitro model, in which fibroblasts are stimulated with transforming growth factor beta 1 (TGF-β1) and stable vitamin C, was used throughout this study. Contractile ability of myofibroblasts was tested using a cell contraction assay. Gene expression of ECM components (collagen I, collagen III, collagen V and lumican), markers of fibrosis (α-smooth muscle actin [α-SMA] and fibronectin), and metalloproteinases (MMP2, MMP9 and MMP12) were assessed by qRT-PCR. Intracellular production and secretion of pro-collagen I and lumican was determined by ELISA. α-SMA protein expression was assessed by western blot.

Results : ACh decreased the contractile ability of the newly formed myofibroblasts. ACh significantly decreased gene expression of collagen I, collagen III and collagen V in myofibroblasts. Moreover, ACh treated cells produced and secreted less pro-collagen I. Gene expression of lumican was unaffected by ACh treatment up to day 2 but significantly decreased by day 4. However, no differences in lumican protein level, both intracellular and secreted, were found. ACh downregulated expression of both α-SMA and fibronectin genes. Additionally, α-SMA protein expression was also diminished in ACh treated cells. Furthermore, ACh treatment resulted in downregulation of MMP2, MMP9 and MMP12 genes.

Conclusions : Our results are consistent with our hypothesis that ACh regulates expression of various collagens, lumican, fibronectin and metalloproteinases during corneal fibrosis in vitro, in a way that it diminishes their expression, both on RNA and protein levels. In conclusion, ACh seems to provide protection against formation of fibrosis in human cornea.

sted, utgiver, år, opplag, sider
The Association for Research in Vision and Ophthalmology, Inc., 2018
HSV kategori
Identifikatorer
urn:nbn:se:umu:diva-152274 (URN)000442912506279 ()
Konferanse
Annual Meeting of the Association-for-Research-in-Vision-and-Ophthalmology (ARVO), APR 29-MAY 03, 2018, Honolulu, HI
Forskningsfinansiär
Swedish Research Council, 521-2013-2612The Kempe Foundations, JCK-1222Swedish Society of Medicine, SLS-410021Swedish Society of Medicine, SLS-410021Västerbotten County Council, VLL-363161Västerbotten County Council, VLL-549761
Tilgjengelig fra: 2018-10-02 Laget: 2018-10-02 Sist oppdatert: 2018-10-02bibliografisk kontrollert
Chen, J., Zhang, W., Backman, L. J., Kelk, P. & Danielson, P. (2018). Mechanical stress potentiates the differentiation of periodontal ligament stem cells into keratocytes. British Journal of Ophthalmology, 102(4), 562-569
Åpne denne publikasjonen i ny fane eller vindu >>Mechanical stress potentiates the differentiation of periodontal ligament stem cells into keratocytes
Vise andre…
2018 (engelsk)Inngår i: British Journal of Ophthalmology, ISSN 0007-1161, E-ISSN 1468-2079, Vol. 102, nr 4, s. 562-569Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Aims To explore the role of corneal-shaped static mechanical strain on the differentiation of human periodontal ligament stem cells (PDLSCs) into keratocytes and the possible synergistic effects of mechanics and inducing medium. Methods PDLSCs were exposed to 3% static dome-shaped mechanical strain in a Flexcell Tension System for 3 days and 7 days. Keratocyte phenotype was determined by gene expression of keratocyte markers. Keratocyte differentiation (inducing) medium was introduced in the Flexcell system, either continuously or intermittently combined with mechanical stimulation. The synergistic effects of mechanics and inducing medium on keratocyte differentiation was evaluated by gene and protein expression of keratocyte markers. Finally, a multilamellar cell sheet was assembled by seeding PDLSCs on a collagen membrane and inducing keratocyte differentiation. The transparency of the cell sheet was assessed, and typical markers of native human corneal stroma were evaluated by immunofluorescence staining. Results Dome-shaped mechanical stimulation promoted PDLSCs to differentiate into keratocytes, as shown by the upregulation of ALDH3A1, CD34, LUM, COL I and COL V. The expression of integrins were also upregulated after mechanical stimulation, including integrin alpha 1, alpha 2, beta 1 and non-muscle myosin II B. A synergistic effect of mechanics and inducing medium was found on keratocyte differentiation. The cell sheets were assembled under the treatment of mechanics and inducing medium simultaneously. The cell sheets were transparent, multilamellar and expressed typical markers of corneal stroma. Conclusion Dome-shaped mechanical stimulation promotes differentiation of PDLSCs into keratocytes and has synergistic effects with inducing medium. Multilamellar cell sheets that resemble native human corneal stroma show potential for future clinical applications.

sted, utgiver, år, opplag, sider
BMJ Publishing Group Ltd, 2018
Emneord
PDLSCs, corneal stroma, mechanics, inducing medium, differentiation, cell-sheet
HSV kategori
Identifikatorer
urn:nbn:se:umu:diva-147334 (URN)10.1136/bjophthalmol-2017-311150 (DOI)000429732500026 ()29306866 (PubMedID)2-s2.0-85044936731 (Scopus ID)
Tilgjengelig fra: 2018-05-17 Laget: 2018-05-17 Sist oppdatert: 2024-07-02bibliografisk kontrollert
Prosjekter
Odlade senceller som en experimentell modell för tendinopati/tendinos - prövande av biokemiska och mekaniska hypoteser för patofysiologin [2009-02921_VR]; Umeå universitetMekanismer för ärrbildning och degeneration i kollagenrik vävnad [2013-02612_VR]; Umeå universitetMekanismer för sårläkning och ärrbildning i ögats hornhinna [2017-01138_VR]; Umeå universitet
Organisasjoner
Identifikatorer
ORCID-id: ORCID iD iconorcid.org/0000-0002-7906-9152