| 1. |
- Mukwaya, Anthony
(författare)
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Regulation of inflammation and angiogenesis in the cornea
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Inflammation and angiogenesis, the growth of new blood vessels from pre-existing ones, are involved in tumor growth, ocular diseases and wound healing. In ocular angiogenesis, new pathological vessels grow into a specific eye tissue, leak fluid, and disrupt vision. The development of safe and effective therapies for ocular angiogenesis is of great importance for preventing blindness, given that current treatments have limited efficacy or are associated with undesirable side effects. The search for alternative treatment targets requires a deeper understanding of inflammation and how it can lead to angiogenesis in the eye in pathologic situations. This thesis provides new insights into the regulation of inflammation and angiogenesis, particularly at the gene expression and phenotypic levels, in different situations characterized by angiogenesis of the cornea, often called corneal neovascularization. For instance, specific genes and pathways are either endogenously activated or suppressed during active inflammation, wound healing, and during resolution of inflammation and angiogenesis, serving as potential targets to modulate the inflammatory and angiogenic response. In addition, as part of the healing response to restore corneal transparency, inflammation and angiogenesis subside with time in the cornea. In this context, LXR/RXR signaling was found to be activated in a time-dependent manner, to potentially regulate resolution of inflammation and angiogenesis. During regression of new angiogenic capillaries, ghost vessels and empty basement membrane sleeves are formed, which can persist in the cornea for a long time. Here, ghost vessels were found to facilitate subsequent revascularization of the cornea, while empty basement membrane sleeves did not revascularize. The revascularization response observed here was characterised by vasodilation, increased inflammatory cell infiltration and by sprouting at the front of the reperfused vessels. Importantly, reactive oxygen species and nitrous oxide signaling among other pro-inflammatory pathways were activated, and at the same time anti-inflammatory LXR/RXR signaling was inhibited. The interplay between activation and inhibition of these pathways highlights potential mechanisms that regulate corneal revascularization. When treating corneal neovascularization clinically, corticosteroids are in widespread use due to their effectiveness. To minimize the many undesirable side effects associated with corticosteroid use, however, identifying new and more selective agents is of great importance. Here, it was observed that corticosteroids not only suppressed pro-inflammatory chemokines and cytokines, but also activated the classical complement pathway. Classical complement may represent a candidate for further selective therapeutic manipulation to investigate its effect on treatment of corneal neovascularization.In summary, this thesis identifies genes, pathways, and phenotypic responses involved in sprouting and remodeling of corneal capillaries, highlights novel pathways and factors that may regulate inflammation and angiogenesis in the cornea, and provides insights into regulation of capillary regression and reactivation. Further investigation of these regulatory mechanisms may offer alternative and effective treatment targets for the treatment of corneal inflammation and angiogenesis.
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| 2. |
- Bourghardt Peebo, Beatrice, 1968-
(författare)
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Angiogenesis from a new perspective
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Angiogenesis is the emergence of new blood and lymph vessels from existing ones. In the pathologic form it contributes to the onset and progression of numerous different human disorders such as cancer, inflammation, atherosclerosis and blinding eye diseases. There exist a number of models to study angiogenesis, both in vitro and in vivo, but there is no single perfect model so far. Consequently there is a need to develop new angiogenesis assays for evaluating blood and lymph vessel behaviour in different physiologic settings.The aim of this thesis was to gain insight into in vivo angiogenesis introducing a new technique in an inflammatory corneal model. The method involved in vivo examination of the cornea and subsequent comparison of in vivo findings with ex vivo immunohistochemical analysis of the same tissue samples. An existing suture model for inflammatory angiogenesis in the cornea was modified for in vivo observations with a clinically-approved corneal confocal microscope.In this thesis, corneal lymph vessels were characterized for the first time in vivo and findings from the experimental bench could be applied in a clinical setting, where presumed lymphatics were observed in a corneal transplant patient with rejection. Furthermore, the technique was extended to investigate time-lapse processes in sprouting and regressing capillaries, and led to a number of new observations. CD11b+ myeloid cells constitute the first bulk of infiltrating inflammatory cells and contribute to inflammatory sprouting and regression in numerous ways including pre-patterning of the corneal stroma and guiding of capillary sprouts. Newly formed hemangiogenic sprouts are perfused with a slow-moving fluid and have a lumen. In blood vessel regression, capillary remodeling occurred by abandonment of sprout tips in close association with macrophages and vascular loops formed by presumed intussusceptive angiogenesis. In addition, a network of pericyte- and endothelium-free basement membrane tubes was formed after desertion or degradation of vascular endothelium in former corneal capillaries.In conclusion, we introduce a new in vivo technique for investigating angiogenesis in a corneal model were in vivo findings can be interpreted with ex vivo definitions of specific cell types by immunohistochemistry. Findings from pre-clinical experiments have been possible to apply in a clinical setting when examining patients with corneal pathology.
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| 3. |
- Germundsson, Johan
(författare)
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Surgical outcomes of phototherapeutic keratectomy on Epithelial basement membrane dystrophy, and the characterisation of Bowman´s Layer
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background. Epithelial basement membrane dystrophy (EBMD) is a common disease of the anterior cornea that can lead to problems with vision and/or painful recurrent erosions of the corneal epithelium. Several treatment options have been used, but recurrence of EBMD after treatment is a problem. Excimer laser phototherapeutic keratectomy (PTK) has become an increasingly popular surgical option in recent years due to its accuracy, reproducibility, and good clinical outcomes. When treating EBMD with PTK, the anterior corneal structures including the epithelium, Bowman´s layer (BL), and subbasal nerves are disrupted or removed completely. Little is known about how BL, nerves, and the stroma recover after PTK treatment, or how they could influence recurrence of EBMD symptoms. Additionally, very little is known about the properties and actual thickness of BL in-vivo.Aims. To improve the understanding and management of EBMD by investigating the clinical diagnosis and treatment of EBMD and its relationship to Bowman´s layer.Method. An excimer laser was used to treat EBMD patients at the Department of Ophthalmology during the period 2001-2010. IVCM was used to perform pre- and postoperative examinations. In particular, images of anterior corneal structures, cells, and nerves in high-resolution were obtained. Additionally, a group of over 100 healthy volunteers underwent a full ophthalmic examination including IVCM. Other subjects examined in this work included a group of 17 patients who underwent full-thickness transplantation of the cornea.Results and conclusions. Clinical follow-up revealed that PTK is an effective method of alleviating the clinical symptoms of EBMD, but the dystrophy can recur with time. Recurrence can be divided into clinical and morphologic types, and may depend upon treatment parameters including the type and depth of ablation. IVCM was found to be a useful screening tool pre- and postoperatively, and could prevent patients with symptoms, but no visible signs of EBMD on slit lamp examination, to go undiagnosed and untreated. BL was found to play a role in regenerative wound healing after PTK, and was also found to be important regarding the treatment and recurrence of EBMD. BL may present a physical barrier that protects the subepithelial nerve plexus thereby facilitating sensory recovery, and BL may also serve as a barrier that prevents direct traumatic contact with the corneal stroma, avoiding a stromal wound healing response. To aid in accurate assessment of BL in patients, an in vivo method for determining BL thickness was developed. This method could be an important tool to aid in clinical assessment and planned treatments of the anterior cornea. Using this tool, a large inter-individual variability in BL thickness and a strong negative correlation of BL thickness with age were found in a healthy population. Using IVCM, it was also found that subbasal nerves are pathologically reduced in EBMD compared to a healthy population, and that this nerve deficit does not improve in the long term after PTK treatment.
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| 4. |
- Kulikovska, Marina
(författare)
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Corneal stromal cell responses to traumatic wounds and topical treatments
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background. The cornea has unique anatomic, cellular, molecular, and functional features that lead to important mechanistic differences in the process of repair in comparison with what occurs in skin and other organs. The first observable stromal response in corneal wound healing is keratocyte apoptosis. Shortly thereafter, remaining keratocytes in adjacent areas obtain a fibroblastic phenotype and begin to proliferate and to migrate, transforming into myofibroblasts, a phenotype associated with remodeling of stromal collagen. Return to normalcy following wound healing includes elimination of myofibroblasts and restoration of the quiescent state of the keratocytes. Often, however, a wound healing response results in the persistence of myofibroblasts and their subsequent production of fibrous scar tissue.Aims. The overall aim is to understand the role of keratocytes, and their phenotypic variations in a cornea subjected to various types of trauma or treatments. More specific aims are to define expression pattern of alpha-smooth muscle actin (α-SMA) and chaperonin containing T-complex polypeptide 1 (CCT) in ultraviolet radiation wound model, to evaluate the effect of biglycan and platelet rich plasma (PRP) treatment during wound healing after corneal incision, and to characterize the structure of the bioengineered porcine construct and its interaction with stromal cells after implantation.Methods. CCT and α-SMA expression level was evaluated by reverse transcription polymerase chain reaction (RT-PCR) in rabbit corneas subjected to ultraviolet radiation (UVR). Effect of biglycan and PRP on keratocyte phenotype and survival was evaluated by immunohistochemistry, and real time PCR using rat corneas after incisional wounding. Bioengineered porcine construct (BPC) was implanted into rabbit corneas using femtosecond laser-enabled intrastromal keratoplasty (FLISK) and characterized by means of immunohistochemistry, electron microscopy, and in vivo confocal microscopy (IVCM).Results and conclusions. In a mild wound, the expression of α-SMA mRNA is followed by expression of mRNA of at least one subunit of the complex folding α-SMA. At protein level, α-SMA is detected in the front line of repopulating keratocytes. Expression levels for both mRNAs decline as the stroma repopulation process progresses.Biglycan appears to accelerate corneal wound healing in vivo by modulating myofibroblast apoptosis, resulting in removal of myofibroblasts that may otherwise compromise corneal transparency.PRP treatment resulted in suppressed stromal cell apoptosis followed by SMAD3 activation and a greater proportion of myofibroblasts present at the wound site. Suppression of stromal cell apoptosis after corneal wounding by use of a growth factor rich formulation may lead to myofibroblast accumulation by modulation of the TGF-β pathway.A cost-effective BPC extracellular matrix equivalent can incorporate cells passively to initiate normal regenerative healing of the corneal stroma.Taken together, results present an interesting possibility to combine BPC implantation and topical biglycan treatment to improve surgical outcome in future studies.
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