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1.
Stem Cells Transl Med ; 9(8): 917-935, 2020 08.
Article in English | MEDLINE | ID: mdl-32379938

ABSTRACT

Corneal scarring associated with various corneal conditions is a leading cause of blindness worldwide. The present study aimed to test the hypothesis that corneal stromal stem cells have a therapeutic effect and are able to restore the extracellular matrix organization and corneal transparency in vivo. We first developed a mouse model of corneal stromal scar induced by liquid nitrogen (N2 ) application. We then reversed stromal scarring by injecting mouse or human corneal stromal stem cells in injured cornea. To characterize the mouse model developed in this study and the therapeutic effect of corneal stromal stem cells, we used a combination of in vivo (slit lamp, optical coherence tomography, in vivo confocal microscopy, optical coherence tomography shear wave elastography, and optokinetic tracking response) and ex vivo (full field optical coherence microscopy, flow cytometry, transmission electron microscopy, and histology) techniques. The mouse model obtained features early inflammation, keratocyte apoptosis, keratocyte transformation into myofibroblasts, collagen type III synthesis, impaired stromal ultrastructure, corneal stromal haze formation, increased corneal rigidity, and impaired visual acuity. Injection of stromal stem cells in N2 -injured cornea resulted in improved corneal transparency associated with corneal stromal stem cell migration and growth in the recipient stroma, absence of inflammatory response, recipient corneal epithelial cell growth, decreased collagen type III stromal content, restored stromal ultrastructure, decreased stromal haze, decreased corneal rigidity, and improved vision. Our study demonstrates the ability of corneal stromal stem cells to promote regeneration of transparent stromal tissue after corneal scarring induced by liquid nitrogen.


Subject(s)
Cell- and Tissue-Based Therapy/methods , Cornea/physiopathology , Stem Cells/metabolism , Animals , Disease Models, Animal , Humans , Mice , Stem Cells/cytology
2.
Front Cardiovasc Med ; 6: 132, 2019.
Article in English | MEDLINE | ID: mdl-31572732

ABSTRACT

Platelets are important actors of cardiovascular diseases (CVD). Current antiplatelet drugs that inhibit platelet aggregation have been shown to be effective in CVD treatment. However, the management of bleeding complications is still an issue in vascular diseases. While platelets can act individually, they interact with vascular cells and leukocytes at sites of vascular injury and inflammation. The main goal remains to better understand platelet mechanisms in thrombo-inflammatory diseases and provide new lines of safe treatments. Beyond their role in hemostasis and thrombosis, recent studies have reported the role of several aspects of platelet functions in CVD progression. In this review, we will provide a comprehensive overview of platelet mechanisms involved in several vascular diseases.

3.
Ann Rheum Dis ; 78(9): 1274-1282, 2019 09.
Article in English | MEDLINE | ID: mdl-31147357

ABSTRACT

OBJECTIVES: Behçet's disease (BD) is a chronic systemic vasculitis. Thrombosis is a frequent and life-threatening complication. The pathogenesis of BD is poorly understood and evidence supporting a role for primed neutrophils in BD-associated thrombotic risk is scant. To respond to inflammatory insults, neutrophils release web-like structures, known as neutrophil extracellular traps (NETs), which are prothrombotic. We evaluated the role of NETs and markers of NETs in BD. METHODS: Blood samples were collected from patients with BD, according to the International Study Group Criteria for Behçet's disease, and healthy donors (HD). NET components, including cell-free DNA (CfDNA) and neutrophil enzymes myeloperoxidase (MPO), were assessed in serum or in purified neutrophils from patients with BD and HD. RESULTS: Patients with active BD had elevated serum cfDNA levels and MPO-DNA complexes compared with patients with inactive BD and to HD. In addition, levels of cfDNA and MPO-DNA complexes were significantly higher in patients with BD with vascular involvement compared with those without vascular symptoms. Purified neutrophils from patients with BD exhibited spontaneous NETosis compared with HD. Thrombin generation in BD plasma was significantly increased and positively correlated with the levels of MPO-DNA complexes and cfDNA. Importantly, DNAse treatment significantly decreased thrombin generation in BD plasma but not in HD plasma. In addition, biopsy materials obtained from patients with BD showed NETs production in areas of vasculitic inflammation and thrombosis. CONCLUSIONS: Our data show that NETs and markers of NETS levels are elevated in patients with BD and contribute to the procoagulant state. Targeting NETs may represent a potential therapeutic target for the reduction or prevention of BD-associated thrombotic risk.


Subject(s)
Behcet Syndrome/blood , Extracellular Traps/metabolism , Neutrophils/metabolism , Adult , Behcet Syndrome/pathology , Biomarkers/blood , Female , Humans , Male , Neutrophils/pathology , Severity of Illness Index
4.
Blood Adv ; 2(18): 2389-2399, 2018 09 25.
Article in English | MEDLINE | ID: mdl-30254103

ABSTRACT

Coagulation and fibrinolytic system deregulation has been implicated in the development of idiopathic pulmonary fibrosis, a devastating form of interstitial lung disease. We used intratracheal instillation of bleomycin to induce pulmonary fibrosis in mice and analyzed the role of serine protease inhibitor E2 (serpinE2)/protease nexin-1 (PN-1), a tissue serpin that exhibits anticoagulant and antifibrinolytic properties. PN-1 deficiency was associated, after bleomycin challenge, with a significant increase in mortality, as well as a marked increase in active thrombin in bronchoalveolar lavage fluids, an overexpression of extracellular matrix proteins, and an accumulation of inflammatory cells in the lungs. Bone marrow transplantation experiments showed that protective PN-1 was derived from hematopoietic cell compartment. A pharmacological strategy using the direct thrombin inhibitor argatroban reversed the deleterious effects of PN-1 deficiency. Concomitant deficiency of the thrombin receptor protease-activated receptor 4 (PAR4) abolished the deleterious effects of PN-1 deficiency in hematopoietic cells. These data demonstrate that prevention of thrombin signaling by PN-1 constitutes an important endogenous mechanism of protection against lung fibrosis and associated mortality. Our findings suggest that appropriate doses of thrombin inhibitors or PAR4 antagonists may provide benefit against progressive lung fibrosis with evidence of deregulated thrombin activity.


Subject(s)
Lung Injury/etiology , Lung Injury/metabolism , Serpin E2/genetics , Serpin E2/metabolism , Signal Transduction , Thrombin/metabolism , Animals , Bleomycin/adverse effects , Blood Cells/metabolism , Blood Coagulation , Disease Models, Animal , Disease Susceptibility , Fibrosis , Lung Injury/mortality , Lung Injury/pathology , Male , Mice , Mice, Knockout , Mice, Transgenic , Receptors, Thrombin/metabolism
5.
PLoS One ; 12(11): e0188398, 2017.
Article in English | MEDLINE | ID: mdl-29149196

ABSTRACT

Epithelial and stromal stem cells are required to maintain corneal transparency. The aim of the study was to develop a new method to isolate and grow both corneal stromal (SSC) and epithelial limbal (LSC) stem cells from small human limbal biopsies under culture conditions in accordance with safety requirements mandatory for clinical use in humans. Superficial limbal explants were retrieved from human donor corneo-scleral rims. Human limbal cells were dissociated by digestion with collagenase A, either after epithelial scraping or with no scraping. Isolated cells were cultured with Essential 8 medium (E8), E8 supplemented with EGF (E8+) or Green's medium with 3T3 feeder-layers. Cells were characterized by immunostaining, RT-qPCR, colony forming efficiency, sphere formation, population doubling, second harmonic generation microscopy and differentiation potentials. LSC were obtained from unscraped explants in E8, E8+ and Green's media and were characterized by colony formation and expression of PAX6, ΔNP63α, Bmi1, ABCG2, SOX9, CK14, CK15 and vimentin, with a few cells positive for CK3. LSC underwent 28 population doublings still forming colonies. SSC were obtained from both scraped and unscraped explants in E8 and E8+ media and were characterized by sphere formation, expression of PAX6, SOX2, BMI1, NESTIN, ABCG2, KERATOCAN, VIMENTIN, SOX9, SOX10 and HNK1, production of collagen fibrils and differentiation into keratocytes, fibroblasts, myofibroblasts, neurons, adipocytes, chondrocytes and osteocytes. SSC underwent 48 population doublings still forming spheres, Thus, this new method allows both SSC and LSC to be isolated from small superficial limbal biopsies and to be primary cultured in feeder-free and xeno-free conditions, which will be useful for clinical purposes.


Subject(s)
Cell Separation/methods , Corneal Stroma/cytology , Epithelial Cells/cytology , Epithelium, Corneal/cytology , Limbus Corneae/cytology , Stem Cells/cytology , ATP Binding Cassette Transporter, Subfamily G, Member 2/genetics , ATP Binding Cassette Transporter, Subfamily G, Member 2/metabolism , Adipocytes/cytology , Adipocytes/drug effects , Adipocytes/metabolism , Biomarkers/metabolism , Cell Differentiation , Cell Proliferation , Chondrocytes/cytology , Chondrocytes/drug effects , Chondrocytes/metabolism , Corneal Stroma/drug effects , Corneal Stroma/metabolism , Culture Media/chemistry , Culture Media/pharmacology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelium, Corneal/drug effects , Epithelium, Corneal/metabolism , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/metabolism , Gene Expression , Humans , Keratinocytes/cytology , Keratinocytes/drug effects , Keratinocytes/metabolism , Limbus Corneae/drug effects , Limbus Corneae/metabolism , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Nestin/genetics , Nestin/metabolism , Neurons/cytology , Neurons/drug effects , Neurons/metabolism , PAX6 Transcription Factor/genetics , PAX6 Transcription Factor/metabolism , Polycomb Repressive Complex 1/genetics , Polycomb Repressive Complex 1/metabolism , Primary Cell Culture , SOX9 Transcription Factor/genetics , SOX9 Transcription Factor/metabolism , Spheroids, Cellular/cytology , Spheroids, Cellular/drug effects , Spheroids, Cellular/metabolism , Stem Cells/drug effects , Stem Cells/metabolism
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