| 1. |
- Chen, Jialin, et al.
(författare)
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Regulation of Keratocyte Phenotype and Cell Behavior by Substrate Stiffness
- 2020
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Ingår i: ACS Biomaterials Science & Engineering. - : American Chemical Society (ACS). - 2373-9878. ; 6:9, s. 5162-5171
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Danielson, Johan, 1975-, et al.
(författare)
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Controlled long term outcome of pyloromyotomy for pyloric stenosis : No long-term adverse effect
- 2022
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Ingår i: Journal of Pediatric Surgery. - : Elsevier. - 0022-3468 .- 1531-5037. ; 57:11, s. 736-739
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: Pyloromyotomy for pyloric stenosis is one of the more common surgical procedures performed on infants. The long-term effects of the procedure are however unclear. The purpose of this study was to study the effects into adult life, compare them with controls and to see if there is a need for structured follow up of patients.Methods: Of the 101 patients operated for pyloric stenosis between 1972 and 1974 at our tertiary referral center 91 could be traced. They were all invited to participate in the study and were sent validated ques-tionnaires (PAGI-SYM, GIQLI) as well as a study-specific questionnaire examining the use of antacid drugs, incidence of gastroscopy and abdominal surgery. Sixty patients responded (66%, mean age 45 years, 46 male) and were included. Thereafter, 600 age and sex-matched controls were sent the same question-naires. 132 responded (22%, 90 male) and were includes as controls.Results: No significant differences could be found in any of the examined parameters when looking at the whole material or the male patients. Female patients had higher PAGI-SYM-scores for post prandial fullness (mean 1.11 vs 0.43, P = 0.035) and heartburn (mean 0.59 vs 0.14, P = 0.043) when compared to controls.Conclusions: The present study shows that most patients operated for pyloric stenosis during infancy experience no negative effects into adulthood. The finding in the female patient group is interesting but is unlikely to have any clinical implications. The results from this study strongly implicate that there is no need for follow up of patients into adulthood.Level of evidence: Level III.
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| 3. |
- El-Habta, Roine, 1988-
(författare)
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Cell therapy for denervated tissue
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background: Peripheral nerve injury results in denervation of tendons and muscles. The biology of denervated muscle has been well studied but little is known about the associated tendons. Denervation of muscle leads to atrophy which includes muscle fiber shrinkage and cell death, a process that is influenced by the lack of acetylcholine (ACh) signaling to the muscle cells. Recovery of long-term denervated muscle function is often poor. This thesis describes how a cell therapy approach using adipose tissue-derived stromal vascular fraction (SVF) may be used to protect and regenerate denervated muscle. Previous studies have shown how adipose tissue-dervied stem cells (ASCs), commonly expanded from the SVF, have pro-regenerative effects on the injured peripheral nervous system, and how ASCs differentiated towards a “Schwann cell-like phenotype” (dASCs) reduce muscle atrophy. In this thesis work, we studied the possible mechanisms underlying the regenerative potential of both SVF and culture expanded dASCs.Hypotheses: We hypothesized that: 1) denervated tendon displays morphological and biochemical properties that resemble the chronic degenerative tendon condition known as tendinosis; 2) denervated muscle up-regulates expression of muscarinic acetylcholine (ACh) receptors and apoptosis-associated signaling mechanisms; 3) dASCs enhance the proliferation of myoblasts in vitro through secretion of ACh; 4) SVF influences the proliferation, differentiation, and survival of myoblasts in vitro via secretion of growth factors; and 5) SVF can preserve denervated muscle tissue. To test our hypotheses, two model systems were used: an in vitro model based on indirect co-culture, and an in vivo rat sciatic nerve transection model.Results: Denervated tendon displayed morphological changes similar to tendinosis, including hypercellularity, disfigurement of cells, and disorganized collagen architecture, along with an increased expression of type I and type III collagen. In addition, levels of neurokinin 1 receptor (NK-1R) were upregulated in the tendon cells. In denervated muscle, there was an increased expression of muscarinic ACh receptors, as well as of genes associated with apoptosis, such as caspases, cytokines (e.g., tumor necrosis factor-alpha; TNF-a), and death domain receptors. We subsequently used TNF-aas an inducer of apoptosis in an in vitrorat primary myoblast culture model. TNF-aactivated/cleaved caspase 7 and increased poly ADP-ribose polymerase (PARP) levels. Moreover, Annexin V and TUNEL were increased after TNF-atreatment. Indirect co-culture with SVF significantly reduced all these measures of apoptosis. Proliferation studies showed that both dASCs and SVF enhanced growth of myoblasts in vitro. With dASCs, the effect was partially explained by secretion of ACh, and for SVF by released growth factors, such as hepatocyte growth factor (HGF). In both cases, the signal was mediated via phosphorylation of ERK1/2 (MAPK). HGF also had an inhibitory effect on the differentiation of myoblasts into myotubes. Finally, the protective effects of SVF were confirmed in vivo: injections of SVF into denervated muscle significantly increased the mean fiber area and diameter, as well as reduced the expression of apoptotic genes and TUNEL reactivity.Conclusions: Denervated tendons undergo severe degenerative changes similar to tendinosis. Furthermore, SVF has the ability to reduce muscle atrophy in vivo. Using in vitro systems, we showed that this might occur through secretion of growth factors which activate MAPK signaling and anti-apoptotic pathways. In conclusion, SVF offers a promising approach for future clinical application in the treatment of denervated muscle.
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| 4. |
- Prittinen, Juha, 1989-, et al.
(författare)
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Microstructured collagen films for 3D corneal stroma modelling
- 2022
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Ingår i: Connective Tissue Research. - : Taylor & Francis Group. - 0300-8207 .- 1607-8438. ; 63:5, s. 443-452
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Sloniecka, Marta, et al.
(författare)
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Acetylcholine decreases formation of myofibroblasts and excessive extracellular matrix production in an in vitro human corneal fibrosis model
- 2020
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Ingår i: Journal of Cellular and Molecular Medicine (Print). - : John Wiley & Sons. - 1582-1838 .- 1582-4934. ; 24:8, s. 4850-4862
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
- Viberg, Andreas, 1984-
(författare)
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Fuchs’ endothelial corneal dystrophy : Genetic aetiology and as a risk factor in cataract surgery
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Fuchs’ endothelial corneal dystrophy (FECD) is a bilateral, often hereditary degenerative corneal disease, in which the disrupted endothelial cell function causes corneal swelling and reduced vision. An early clinical sign of FECD is corneal guttata, an irregularity of the endothelial layer. Ocular surgery, not least cataract surgery, can lead to endothelial damage and progression of FECD. In Sweden and most Western countries, FECD is the most common indication for corneal transplantation. It has been considered a genetically heterogeneous disease but is increasingly associated with a (CTG)n repeat expansion in transcription factor 4 gene (TCF4). In this thesis, the association between (CTG)n repeat expansion in TCF4 and FECD was investigated, and it was hypothesised that the repeat length correlates with disease severity. The results show that FECD in Northern Sweden is associated with (CTG)n expansion in the TCF4 gene to a large extent. More precisely, expansion with more than 50 repeats in TCF4 was present in nearly 90% of the 85 tested FECD cases compared to only 3.9% in controls, which is the highest published prevalence to date. The disease's severity was associated with the repeat length in the TCF4 gene with an adjusted odds ratio (OR) of 2.13 (95% CI, 1.34-3.39) per repeat length unit. Further, we studied the impact of corneal guttata on cataract surgery outcome, using the data from nationwide eye registries. We show that patients with corneal guttata/FECD benefit from cataract surgery with improved visual acuity and self-assessed visual function, but that they have a greater risk of corneal transplantation and worse results of the cataract surgery than patients without FECD. The risk of corneal transplantation after cataract surgery in patients with corneal guttata was 68 times higher than in patients without corneal guttata. The risk was highest the first year after cataract surgery and decreased after that. Complicated cataract surgery with a dense lens and posterior capsule rupture, both individually and together, increased the risk of corneal transplantation, independent of corneal guttata. In conclusion, most patients with FECD and concomitant symptomatic cataract benefit from cataract surgery. It is not surprising that the risk of corneal transplantation after cataract surgery is increased in patients with FECD, as FECD is an indication for corneal transplantation. Still, the vast majority of FECD patients do not undergo a corneal transplant after cataract surgery. With the results of this thesis as a basis, we recommend, to start with cataract surgery before planning for corneal transplantation in most cases of FECD. Additionally, the surgery should be performed before the lens becomes hazardly dense and with caution to minimise the risk of posterior capsule rupture.
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| 7. |
- Zhang, Wei, et al.
(författare)
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Sustained Release of TPCA-1 from Silk Fibroin Hydrogels Preserves Keratocyte Phenotype and Promotes Corneal Regeneration by Inhibiting Interleukin-1β Signaling
- 2020
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Ingår i: Advanced Healthcare Materials. - : Wiley-VCH Verlagsgesellschaft. - 2192-2640 .- 2192-2659. ; 9:17
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 8. |
- Zhou, Xin, et al.
(författare)
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Activation of NF-κB signaling via cytosolic mitochondrial RNA sensing in kerotocytes with mitochondrial DNA common deletion
- 2021
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 9. |
- Zhou, Xin, et al.
(författare)
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Keratocyte Differentiation Is Regulated by NF-κB and TGFβ Signaling Crosstalk
- 2022
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Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 23:19
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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