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  • 1.
    Borbely, Gabor
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Löfgren, Filip
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Sloniecka, Marta
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    The role of neurokinin A in corneal wound repair2015In: Investigative Ophthalmology and Visual Science, ISSN 0146-0404, E-ISSN 1552-5783, Vol. 56, no 7, article id Meeting Abstract: 725Article in journal (Other academic)
  • 2.
    El-Habta, Roine
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Sloniecka, Marta
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Kingham, Paul J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    The adipose tissue stromal vascular fraction secretome enhances the proliferation but inhibits the differentiation of myoblasts2018In: Stem Cell Research & Therapy, E-ISSN 1757-6512, Vol. 9, article id 352Article in journal (Refereed)
    Abstract [en]

    Background: Adipose tissue is an excellent source for isolation of stem cells for treating various clinical conditions including injuries to the neuromuscular system. Many previous studies have focused on differentiating these adipose stem cells (ASCs) towards a Schwann cell-like phenotype (dASCs), which can enhance axon regeneration and reduce muscle atrophy. However, the stromal vascular fraction (SVF), from which the ASCs are derived, also exerts broad regenerative potential and might provide a faster route to clinical translation of the cell therapies for treatment of neuromuscular disorders.

    Methods: The aim of this study was to establish the effects of SVF cells on the proliferation and differentiation of myoblasts using indirect co-culture experiments. A Growth Factor PCR Array was used to compare the secretomes of SVF and dASCs, and the downstream signaling pathways were investigated.

    Results: SVF cells, unlike culture-expanded dASCs, expressed and secreted hepatocyte growth factor (HGF) at concentrations sufficient to enhance the proliferation of myoblasts. Pharmacological inhibitor studies revealed that the signal is mediated via ERK1/2 phosphorylation and that the effect is significantly reduced by the addition of 100 pM Norleual, a specific HGF inhibitor. When myoblasts were differentiated into multinucleated myotubes, the SVF cells reduced the expression levels of fast-type myosin heavy chain (MyHC2) suggesting an inhibition of the differentiation process.

    Conclusions: In summary, this study shows the importance of HGF as a mediator of the SVF effects on myoblasts and provides further evidence for the importance of the secretome in cell therapy and regenerative medicine applications.

  • 3.
    Erttmann, Saskia F.
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Härtlova, Anetta
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Sloniecka, Marta
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Raffi, Faizal A. M.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Hosseinzadeh, Ava
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Edgren, Tomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Rofougaran, Reza
    Resch, Ulrike
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Fällman, Maria
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Ek, Torben
    Gekara, Nelson O.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Loss of the DNA Damage Repair Kinase ATM Impairs Inflammasome-Dependent Anti-Bacterial Innate Immunity2016In: Immunity, ISSN 1074-7613, E-ISSN 1097-4180, Vol. 45, no 1, p. 106-118Article in journal (Refereed)
    Abstract [en]

    The ATM kinase is a central component of the DNA damage repair machinery and redox balance. ATM dysfunction results in the multisystem disease ataxia-telangiectasia (AT). A major cause of mortality in AT is respiratory bacterial infections. Whether ATM deficiency causes innate immune defects that might contribute to bacterial infections is not known. Here we have shown that loss of ATM impairs inflammasome- dependent anti-bacterial innate immunity. Cells from AT patients or Atm(-/-) mice exhibited diminished interleukin-1 beta (IL-1 beta) production in response to bacteria. In vivo, Atm(-/-) mice were more susceptible to pulmonary S. pneumoniae infection in a manner consistent with inflammasome defects. Our data indicate that such defects were due to oxidative inhibition of inflammasome complex assembly. This study reveals an unanticipated function of reactive oxygen species (ROS) in negative regulation of inflammasomes and proposes a theory for the notable susceptibility of AT patients to pulmonary bacterial infection.

  • 4.
    Roux, Sandrine Le
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Borbely, Gabor
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Słoniecka, Marta
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
    Backman, Ludvig J
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
    Transforming Growth Factor Beta 1 Modulates the Functional Expression of the Neurokinin-1 Receptor in Human Keratocytes2016In: Current Eye Research, ISSN 0271-3683, E-ISSN 1460-2202, Vol. 41, no 8, p. 1035-1043Article in journal (Refereed)
    Abstract [en]

    PURPOSE: Transforming growth factor beta 1 (TGF-β1) is a cytokine involved in a variety of processes, such as differentiation of fibroblasts into myofibroblasts. TGF-β1 has also been shown to delay the internalization of the neurokinin-1 receptor (NK-1 R) after its activation by its ligand, the neuropeptide substance P (SP). NK-1 R comprises two naturally occurring variants, a full-length and a truncated form, triggering different cellular responses. SP has been shown to affect important events in the cornea - such as stimulating epithelial cell proliferation - processes that are involved in corneal wound healing and thus in maintaining the transparency of the corneal stroma. An impaired signaling through NK-1 R could thus impact the visual quality. We hypothesize that TGF-β1 modulates the expression pattern of NK-1 R in human corneal stroma cells, keratocytes. The purpose of this study was to test that hypothesis.

    METHODS: Cultures of primary keratocytes were set up with cells derived from healthy human corneas, obtained from donated transplantation graft leftovers, and characterized by immunocytochemistry and Western blot. Immunocytochemistry for TGF-β receptors and NK-1 R was performed. Gene expression was assessed with real-time polymerase chain reaction (qPCR).

    RESULTS: Expression of TGF-β receptors was confirmed in keratocytes in vitro. Treating the cells with TGF-β1 significantly reduced the gene expression of NK-1 R. Furthermore, immunocytochemistry for NK-1 R demonstrated that it is specifically the expression of the full-length isotype of the receptor that is reduced after treatment with TGF-β1, which was also confirmed with qPCR using a specific probe for the full-length receptor.

    CONCLUSIONS: TGF-β1 down-regulates the gene expression of the full-length variant of NK-1 R in human keratocytes, which might impact its signaling pathway and thus explain the known delay in internalization after activation by SP seen with TGF-β1 treatment.

  • 5.
    Sloniecka, Marta
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Department of Clinical Sciences.
    Neuropeptides and neurotransmitters in keratocytes: importance in corneal wound healing processes2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Background: The cornea is the outermost transparent layer of the eye and it is responsible for the majorityof the eye’s total focusing power. Keratocytes are the resident cells of the corneal stroma and their function isto produce extracellular matrix components and to take part in corneal healing after injury, which may occurdue to trauma, infection or surgery. The process of corneal wound healing is complex. Shortly, keratocytesadjacent to the corneal wound undergo apoptosis and remaining cells start the process of proliferation andmigration in order to close the wound. Next, an influx of inflammatory cells such as macrophages andneutrophils occurs in order to clear the cornea from cellular debris. The final stage of the healing processrestores the quiescent state of keratocytes and remodels any disordered extracellular matrix components,leading to a healthy, transparent cornea. However, when the process of corneal wound healing is incompleteor disturbed, corneal scarring may occur, which can lead to significantly impaired vision. Despite extensiveresearch on corneal wound healing, corneal scarring remains a major cause of preventable blindness. Thehealing process is dependent on various cytokines and growth factors. However, it is possible that also othersignal substances are involved. Substance P (SP) is a neuropeptide well known for its role in pain perception.It has been shown that SP can also be produced by non-neuronal cells, including cells of the cornea, and thatit can have vast effects on physiological functions, including immune cell activity, and cellular processes, suchas cell migration, proliferation, and production of proinflammatory cytokines. Similarly, acetylcholine (ACh),a classical neurotransmitter, has also been reported to be produced by non-neuronal cells, including cornealepithelium, and to be involved in cell proliferation, angiogenesis, cell migration, apoptosis, and collagen geneexpression. In the studies of this thesis, it is hypothesized that neuropeptides and neurotransmitters areproduced by human keratocytes and that this production is increased in response to corneal injury. Moreover,it is hypothesized that the non-neuronal SP and ACh produced by injured keratocytes participate in cornealwound healing by enhancing keratocyte migration and proliferation, and/or by decreasing keratocyteapoptosis. The aims of this thesis project were to test these hypotheses and to study the underlying inter- andintracellular mechanisms of the effects of SP and ACh on keratocytes.Results: Cultured primary cells of the human corneal stroma expressed keratocyte markers (keratocan,lumican, CD34, and ALDH), the tachykinins SP and NKA, catecholamines (adrenaline, noradrenaline anddopamine), ACh, and glutamate. Moreover, the cells expressed neurokinin-1 and -2 receptors (NK-1R andNK-2R), dopamine receptor D2, muscarinic ACh receptors (mAChRs) M1, M3, M4 and M5, and NDMAR1glutamate receptor. Significant differences were observed between expression profiles in cultured keratocytesobtained from central and peripheral cornea. Such differences could also be seen between keratocytescultured under various serum concentrations. Expression and secretion of SP in cultured keratocytes wasincreased in response to injury in vitro. SP enhanced migration of cultured keratocytes through stimulation ofits preferred receptor, the NK-1R, and activation of the phosphatidylinositide 3-kinase and Rac1/RhoApathway and subsequent actin cytoskeleton reorganization and formation of focal adhesion points. Moreover,SP stimulation led to upregulated expression of the proinflammatory and chemotactic cytokine interleukin-8(IL-8), which also contributed significantly to SP-enhanced keratocyte migration and to attractingneutrophils. ACh enhanced keratocyte proliferation in vitro at low concentrations and this stimulation wasmediated through activation of mAChRs and activation of MAPK signalling. Moreover, ACh stimulation led toupregulation of two proliferation markers: PCNA and Ki-67. ACh was also able to protect cultured keratocytesfrom Fas-induced apoptosis, even at low concentrations. Activation of mAChRs was necessary for this latterprocess to occur. ACh reduced caspases 3/7 activation in Fas-treated keratocytes. Inhibition of the PKB/Aktpathway revealed that its activation is essential for mediating the anti-apoptotic effect of ACh in keratocytes.Conclusions: This thesis shows that human keratocytes express an array of neuropeptides (SP, NKA) andneurotransmitters (ACh, adrenaline, noradrenaline, dopamine and glutamate), and their receptors, and thatstimulation of NK-1R by SP and stimulation of mAChRs by ACh lead to keratocyte cellular processes that areknown to be involved in corneal wound healing. Specifically, SP enhances keratocyte migration throughupregulation of IL-8, ACh enhances keratocyte proliferation through activation of the MAPK signallingpathway, and ACh is able to protect keratocytes from apoptosis by activation of the PKB/Akt pathway. Takentogether, these findings suggest that both SP and ACh, if entered at the proper stage, could be beneficial forcorneal wound healing.

  • 6.
    Sloniecka, Marta
    et al.
    Umeå University.
    Backman, L.J
    Chen, J
    Danielsson, P
    Acetylcholine has an anti-apoptotic effect on Fas-induced apoptosis in human keratocytesManuscript (preprint) (Other academic)
  • 7.
    Sloniecka, Marta
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Acetylcholine induces proliferation of keratocytes through activation of muscarinic receptors2015In: Investigative Ophthalmology and Visual Science, ISSN 0146-0404, E-ISSN 1552-5783, Vol. 56, no 7, article id 723Article in journal (Other academic)
    Abstract [en]

    Purpose: The corneal wound healing response is a complex process involving cytokine-mediated interactions between epithelial cells, keratocytes of the stroma, corneal nerves, tear film, and cells of the immune system. The outcome of the response plays a critical role in the preservation of corneal transparency after injury. The wound healing cascade includes epithelial surface closure, keratocyte apoptosis, proliferation and migration, formation of myofibroblasts, and stromal remodeling.<br /> Acetylcholine (ACh) is regarded as a classical neurotransmitter. However, cells outside of the nervous system have been shown to contain and release ACh. It has been reported that ACh stimulates fibroblast and epithelial cells to proliferate, has an anti-inflammatory effect in macrophages, and upregulates collagen gene expression in fibroblasts. ACh, its muscarinic receptors (mAChRs) and choline acetyltransferase (ChAT; the enzyme responsible for synthesizing ACh) have been shown to be present in corneal epithelium. However, their role in the corneal stroma and corneal injury has not been extensively studied.<br /> We hypothesize that ACh, upon injury, induces corneal stroma cell proliferation, thus promoting the process of wound healing.

    Methods: Primary human corneal stroma cells were derived from healthy corneas obtained from the local cornea bank. Immunocytochemistry was performed to delineate intracellular presence of ChAT and to characterize mAChRs. Crystal violet and MTS assay were used to asses ACh induced cell proliferation. Expression of the proliferation markers PCNA and Ki67 was analyzed by western blot. To determine what type of ACh receptors are involved in ACh induced proliferation, atropine and mecamylamine were used to block muscarininc or nicotinic ACh receptors, respectively.

    Results: Stromal cells expressed ChAT as well as mAChRs of subtypes M1, M3, M4, and M5. Stimulation of stromal cells with ACh led to increased cell viability and metabolic activity. Expression of PCNA and Ki67 was upregulated in ACh treated cells. Furthermore, mAChRs were the receptor group primarily involved in ACh induced proliferation.

    Conclusions: Corneal stroma cells express ChAT and mAChRs. ACh induces stroma cell proliferation through mainly mAChRs, which suggest that ACh may play an important role in corneal wound healing i.e. wound closure and generation on myofibroblasts.

  • 8.
    Sloniecka, Marta
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
    Antiapoptotic Effect of Acetylcholine in Fas-Induced Apoptosis in Human Keratocytes2016In: Investigative Ophthalmology and Visual Science, ISSN 0146-0404, E-ISSN 1552-5783, Vol. 57, no 14, p. 5892-5902Article in journal (Refereed)
    Abstract [en]

    PURPOSE. To investigate the possible antiapoptotic effect of acetylcholine (ACh) in Fas-mediated apoptosis of primary human keratocytes in vitro, and to explore the underlying mechanism. METHODS. Primary human keratocytes were isolated from healthy corneas. Fas ligand (FasL) was used to induce apoptosis in keratocytes. Cell death was assessed by ELISA. Activity of caspase-3, -7, -8, and -9 was measured with luminescent caspase activity assays. Expression of nuclear factor-kappa B (NF-kappa B) gene was assessed with RT-quantitative (q)PCR. Cytochrome c release apoptosis assay kit was used to extract mitochondria and cytosol. Cytochrome c release, cleavage of Bid, and expression of B-cell lymphoma 2 (Bcl-2) were determined by Western blot. RESULTS. Cell death ELISA revealed that ACh is able to reduce Fas-induced apoptosis in keratocytes. Analysis of the activity of effector caspases-3 and -7 showed that ACh, when added to Fas-treated cells, decreases the activation of both these enzymes. The activity of initiator caspases -8 and -9 also decreased when ACh was added to Fas-treated cells. This antiapoptotic effect of ACh was dependent on ACh concentration and activation of muscarinic ACh receptors. Analysis of the antiapoptotic mechanisms triggered by ACh showed that ACh downregulates expression of FasL-induced NF-kappa B RNA expression, upregulates expression of antiapoptotic protein Bcl-2, downregulates expression of proapoptotic protein Bad, reduces cytochrome c release, and prevents proapoptotic Bid protein cleavage. CONCLUSIONS. Acetylcholine has an antiapoptotic effect in a Fas-apoptosis model of human primary keratocytes in vitro. It is therefore possible that ACh may play a role in corneal wound healing, by modulating its initiation phase.

  • 9.
    Sloniecka, Marta
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Acetylcholine decreases formation of myofibroblasts and excessive extracellular matrix components production in an in vitro human corneal fibrosis model2018In: Investigative Ophthalmology and Visual Science, ISSN 0146-0404, E-ISSN 1552-5783, Vol. 59, no 9Article in journal (Other academic)
    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.

  • 10.
    Sloniecka, Marta
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology. Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Substance P induces fibrotic changes through activation of the RhoA/ROCK pathway in an in vitro human corneal fibrosis model2019In: Journal of Molecular Medicine, ISSN 0946-2716, E-ISSN 1432-1440, Vol. 97, no 10, p. 1477-1489Article in journal (Refereed)
    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.

  • 11.
    Sloniecka, Marta
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Substance P Promotes Fibrosis in Human Corneal Stroma2018In: Wound Repair and Regeneration, ISSN 1067-1927, E-ISSN 1524-475X, Vol. 26, no 1, p. A22-A22, article id N4.05Article in journal (Other academic)
  • 12.
    Sloniecka, Marta
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Le Roux, Sandrine
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Boman, Peter
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Byström, Berit
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
    Zhou, Qingjun
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Shandong Provincial Key Laboratory of Ophthalmology, Shandong Eye Institute, Qingdao, China.
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
    Expression Profiles of Neuropeptides, Neurotransmitters, and Their Receptors in Human Keratocytes In Vitro and In Situ2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 7, article id e0134157Article in journal (Refereed)
    Abstract [en]

    Keratocytes, the quiescent cells of the corneal stroma, play a crucial role in corneal wound healing. Neuropeptides and neurotransmitters are usually associated with neuronal signaling, but have recently been shown to be produced also by non-neuronal cells and to be involved in many cellular processes. The aim of this study was to assess the endogenous intracellular and secreted levels of the neuropeptides substance P (SP) and neurokinin A (NKA), and of the neurotransmitters acetylcholine (ACh), catecholamines (adrenaline, noradrenaline and dopamine), and glutamate, as well as the expression profiles of their receptors, in human primary keratocytes in vitro and in keratocytes of human corneal tissue sections in situ. Cultured keratocytes expressed genes encoding for SP and NKA, and for catecholamine and glutamate synthesizing enzymes, as well as genes for neuropeptide, adrenergic and ACh (muscarinic) receptors. Keratocytes in culture produced SP, NKA, catecholamines, ACh, and glutamate, and expressed neurokinin-1 and -2 receptors (NK-1R and NK-2R), dopamine receptor D-2, muscarinic ACh receptors, and NDMAR1 glutamate receptor. Human corneal sections expressed SP, NKA, NK-1R, NK-2R, receptor D2, choline acetyl transferase (ChAT), M-3, M4 and M-5 muscarinic ACh receptors, glutamate, and NMDAR1, but not catecholamine synthesizing enzyme or the alpha(1) and beta(2) adrenoreceptors, nor M1 receptor. In addition, expression profiles assumed significant differences between keratocytes from the peripheral cornea as compared to those from the central cornea, as well as differences between keratocytes cultured under various serum concentrations. In conclusion, human keratocytes express an array of neuropeptides and neurotransmitters. The cells furthermore express receptors for neuropeptides/neurotransmitters, which suggests that they are susceptible to stimulation by these substances in the cornea, whether of neuronal or non-neuronal origin. As it has been shown that neuropeptides/neurotransmitters are involved in cell proliferation, migration, and angiogenesis, it is possible that they play a role in corneal wound healing.

  • 13.
    Sloniecka, Marta
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
    Le Roux, Sandrine
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Zhou, Qingjun
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Shandong Provincial Key Laboratory of Ophthalmology, Shandong Eye Institute, Qingdao, China.
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Substance P Enhances Keratocyte Migration and Neutrophil Recruitment through Interleukin-82016In: Molecular Pharmacology, ISSN 0026-895X, E-ISSN 1521-0111, Vol. 89, no 2, p. 215-225Article in journal (Refereed)
    Abstract [en]

    Keratocytes, the resident cells of the corneal stroma, are responsible for maintaining turnover of this tissue by synthesizing extracellular matrix components. When the cornea is injured, the keratocytes migrate to the wounded site and participate in the stromal wound healing. The neuropeptide substance P (SP), which is also known to be produced by non-neuronal cells, has previously been implicated in epithelial wound healing after corneal injury. Corneal scarring, which occurs in the stroma when the process of wound healing has malfunctioned, is one of the major causes of preventable blindness. This study aimed to elucidate the potential role of SP in keratocyte migration and therefore in stromal wound healing. We report that the expression and secretion of SP in human keratocytes are increased in response to injury in vitro. Moreover, SP enhances the migration of keratocytes by inducing the actin cytoskeleton reorganization and focal adhesion formation through the activation of the phosphatidylinositide 3-kinase and Ras-related C3 botulinum toxin substrate 1/Ras homolog gene family, member A pathway. Furthermore, SP stimulation leads to upregulated expression of the proinflammatory and chemotactic cytokine interleukin-8 (IL-8), which also contributes significantly to SP-enhanced keratocyte migration and is able to attract neutrophils. In addition, the preferred SP receptor, the neurokinin-1 receptor, is necessary to induce keratocyte migration and IL-8 secretion. In conclusion, we describe new mechanisms by which SP enhances migration of keratocytes and recruits neutrophils, two necessary steps in the corneal wound-healing process, which are also likely to occur in other tissue injuries.

  • 14.
    Sloniecka, Marta U.
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Acetylcholine regulates expression of lumican and collagen i in keratocytes in quiescent state and after transitioning to fibroblasts and myofibroblasts2017In: Wound Repair and Regeneration, ISSN 1067-1927, E-ISSN 1524-475X, Vol. 25, no 4, p. A32-A32Article in journal (Other academic)
  • 15.
    Sloniecka, Marta U.
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Le Roux, S.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Substance P enhances keratocyte migration through activation of the phosphoinositide 3-kinase-Rac1/RhoA pathway2015In: Wound Repair and Regeneration, ISSN 1067-1927, E-ISSN 1524-475X, Vol. 23, no 2, p. A40-A40Article in journal (Other academic)
  • 16.
    Słoniecka, Marta
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Backman, Ludvig J
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Acetylcholine enhances keratocyte proliferation through muscarinic receptor activation.2015In: International Immunopharmacology, ISSN 1567-5769, E-ISSN 1878-1705, Vol. 29, no 1, p. 57-62Article in journal (Refereed)
    Abstract [en]

    Acetylcholine (ACh), a classical neurotransmitter, has been shown to be present in various non-neuronal cells, including cells of the eye, such as corneal epithelium and endothelium, and to have widespread physiological effects such as cytoskeleton reorganization, cellular proliferation, differentiation, and apoptosis. The aim of this study was to investigate the effect of ACh on corneal keratocyte proliferation, and the underlying mechanisms, in order to explore its possible effect in corneal wound healing. Primary culture of human keratocytes was established from donated corneas. Cell viability and fraction of proliferating cells were detected by MTS assay and BrdU incorporation ELISA, respectively. Expression of proliferative markers, PCNA and Ki-67, was detected by western blot and immunocytochemistry. Activation of the MAPK/Erk signaling pathway and its involvement in ACh-enhanced proliferation was determined by western blot analysis, MTS, and BrdU ELISA. We found that ACh enhanced keratocyte proliferation even at low concentrations. Stimulation of proliferation was mediated through activation of muscarinic ACh receptors (mAChRs). Western blot analysis revealed that ACh stimulation of keratocytes upregulated the expression of PCNA and Ki-67, and Ki-67 immunocytochemistry showed that ACh-treated cells were in an active phase of the cell cycle. ACh activated MAPK signaling, and this step was crucial for the ACh-enhanced proliferation, as inhibition of the MAPK pathway resulted in ACh having no proliferative effect. In conclusion, ACh enhances keratocyte proliferation and might thus play a role in proper corneal wound healing.

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