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- 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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| 2. |
- Bourghardt Peebo, Beatrice, 1968-, et al.
(författare)
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Expression of the focal adhesion protein PINCH in normal and alkali-injured corneas and the role of PMNs
- 2007
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Ingår i: Acta Ophthalmologica Scandinavica. - : Wiley. - 1395-3907 .- 1600-0420. ; 85:4, s. 395-400
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: To evaluate the role of particularly interesting new cysteine-histidine-rich protein (PINCH) in corneal wound healing and early neovascularization and to assess the influence of granulocytes. Methods: A standardized corneal alkali wound was inflicted under general anaesthesia to the right eye of 14 New Zealand White rabbits. Seven of the rabbits received i.v. 5 mg/kg fucoidin every 2 hours to prevent granulocytes from entering the wound area. After 36 hours, the rabbits were killed, the corneas excised, fixed in 4% formaldehyde and embedded in paraffin. The sections were double-stained with antibodies against PINCH and with haematoxylin. Results: In the normal cornea and limbus, PINCH was weakly expressed in the corneal epithelium and in a wedge of the conjunctival stroma. In the wounded corneas, PINCH expression was seen in the frontline of repopulating endothelial and epithelial cells, and in active keratocytes. The vascular endothelium and the granulocytes expressed PINCH, as did the conjunctival epithelium. In the fucoidin-treated rabbits, PINCH expression was markedly reduced. The vascular endothelial cells and the few granulocytes did not express PINCH in these rabbits. Conclusions: PINCH is only slightly expressed in the normal cornea. A corneal wound induces PINCH expression in the repopulating cells, in the vascular endothelial cells of the limbus, in the limbal epithelium and in the granulocytes. Exclusion of granulocytes reduces expression of PINCH and there is no expression at all in the vascular endothelium. © 2007 The Authors Journal compilation 2007 Acta Ophthalmol Scand.
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| 5. |
- Mirabelli, Pierfrancesco
(författare)
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Inhibitors of corneal inflammation and angiogenesis : Prospectives and challenges
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Pathologic angiogenesis is involved in cancer and several blinding conditions such as wet age-related macular degeneration, proliferative retinopathies and corneal neovascularization.In these dieseases, the angiogenic triggers are hypoxia and inflammation, and both involve the main angiogenic mediator, which is Vascular Endothelial Growth Factor (VEGF). Among available treatments, anti-VEGF often shows limited or temporary efficacy, while steroids are potentially responsible for many side-effects. This thesis presents a series of linked studies aimed at elucidating the early pathologic changes leading to inflammation and corneal neovascularization, and how various treatments affect this process. In this thesis, anti-inflammatory and anti-angiogenic treatments are applied in corneal neovascularization models, to identify VEGF-independent pathways and other novel factors as future therapy targets, as well as to investigate the endogenous modulation of angiogenesis.A model of experimental neovascularization in the rat cornea was used as main model, where the neovascular response is triggered by a surgical suture placed into the cornea. Investigational treatments (anti-Vegf, dexamethasone, IMD0354, Gap27, or control substances) were then given topically, with the exception of IMD0354, which was given systemically. The effects in the cornea were studied in vivo with slit lamp photography to assess and quantify macroscopic vessel growth and using in vivo confocal microscopy (IVCM) to study cell infiltration and limbal vessel dilation and detect microscopic vessel sprouts; these examinations were performed longitudinally. Genomic analysis with RNA microarray, selected gene expression with q-RT-PCR, and selected protein expression in tissue (immunohistochemistry, immunofluorescence, Western blot) were performed at different time-points. Moreover, other experiments on cell cultures (HUVEC and HCEC), organ cultures (human corneas), ex vivo models (aortic rings) and in vivo studies (zebrafish vasculogenesis) were performed.Dexamethasone suppressed limbal vasodilation and corneal neovascularization more than anti-Vegf, despite no difference in inflammatory cell infiltration into the cornea. Five-hundred eleven fewer genes were differentially expressed in dexamethasone-treated corneas relative to naïve corneas, compared to anti-Vegf. Among them, several major pro-angiogenic and pro-inflammatory factors and chemokines were suppressed only by dexamethasone and represent novel candidate factors to target in order to improve anti-VEGF treatment. On the other hand, selective inhibition of a single inflammatory pathway (NF-κB), despite showing similar early effects as dexamethasone in suppressing tissue inflammation, was not effective enough to suppress new vessel growth. The same factors suppressed by dexamethasone are also inhibited in endogenous modulation of angiogenesis. Surprisingly, dexamethasone activated several complement factors, which could possibly be beneficial in the anti-angiogenic response.In a different therapeutic approach, promoting cell migration to accelerate epithelial wound closure similarly was not sufficient to avoid inflammation and angiogenesis in the cornea.In conclusion, new and more effective treatments are needed for corneal inflammation and neovascularization with fewer side-effects. In this thesis, several novel factors and mechanisms related to inflammation are identified, factors that are not addressed by anti-Vegf therapy, and therefore represent interesting objects for further study, as they have the potential to be targets for adjuvant therapy. Specific anti-inflammatory treatment as well as therapeutic activation of endogenous regulatory pathways, and potentially complement modulation, might represent new strategies to improve anti-angiogenic therapy, but when used alone they do not seem to avoid corneal neovascularization.
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| 6. |
- 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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| 7. |
- Wiklund, Anna, et al.
(författare)
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ACUTE EXUDATIVE POLYMORPHOUS VITELLIFORM MACULOPATHY IN A YOUNG WOMAN: PRESYMPTOMATIC FINDINGS AND 21-MONTH FOLLOW-UP
- 2013
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Ingår i: RETINAL Cases & Brief Reports. - 1935-1089. ; 7:2, s. 123-127
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: To describe ocular findings before and after the diagnosis of acute exudative polymorphous vitelliform maculopathy, in an otherwise healthy 28-year-old woman.Methods: Case report with 21-months of follow-up. Fundus photography, optical coherence tomography, fluorescein angiography, indocyanine green angiography, and autofluorescence were used for imaging the retina. To examine retinal function, full-field electroretinogram, multifocal electroretinogram, electrooculography, and dark adaptometry were performed. Genetic analysis for mutations associated with Best disease was done.Results: In the asymptomatic patient before diagnosis, white-yellow, drusen-like, subretinal depositions were found in both eyes. A few months later, the patient developed bilateral visual disturbances. Retinal examination at the acute phase revealed a characteristic pattern of multifocal white-yellow subretinal lesions in both posterior poles, imaged by ophthalmoscopy, fluorescein angiography, indocyanine green angiography, and optical coherence tomography. Additionally, electrooculography and dark adaptometry were abnormal. Full-field electroretinogram was normal, but multifocal electroretinogram revealed central depression of peak amplitudes. During the 21-month follow-up without any treatment, visual acuity recovered, electrooculography and dark adaptometry normalized, and the patient experienced one episode of relapse. Genetic studies excluded mutations in the bestrophin gene (BEST1).Conclusion: Acute exudative polymorphous vitelliform maculopathy is still a condition of unknown origin, primarily affecting the pigment epithelium. Earlier reports have discussed whether the condition is inherited or acquired. In this report, the presymptomatic retinal findings in acute exudative polymorphous vitelliform maculopathy are described for the first time, indicating that a condition may be associated with primarily affected retinal pigment epithelium.
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| 8. |
- Xeroudaki, Maria, 1989-
(författare)
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Advanced surgeries, medicines and materials for corneal regeneration
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Corneal transplantation is often the only treatment option in cases of corneal blindness, with the main challenges being the scarcity of human donors, risk for graft failure and suboptimal visual outcome due to suture-related issues. Alternative therapies are sought that either address the above transplantation issues directly or stimulate the cornea’s repair mechanism and regenerative properties to restore transparency without the need for transplantation. The first aim of this thesis was to develop a cell-free substitute for the human corneal stroma made of porcine collagen, a purified byproduct from the food industry that is already approved by FDA as a raw material, used for example in cosmetic surgery or as a medical device in glaucoma surgery. Abundance, cost-effectiveness, low rejection risk due to acellularity, high purity and worldwide availability are among the main advantages of purified porcine collagen compared to human donors and other corneal stromal substitutes. The second aim was to address the risk of graft rejection in cases of neovascularized and inflamed corneas by loading the bioengineered porcine collagen constructs with drugs that can ideally promote corneal regeneration, secure the survival of the graft in cases of high-risk keratoplasties and minimize the need for prolonged topical immunosuppressive therapy following operation, which has drawbacks of low bioavailability and need for good patient compliance. The third aim was to develop alternative techniques of lamellar transplantation, all assisted by femtosecond laser, that are less suture-dependent and enable the intrastromal implantation of biomaterials. Finally, the fourth aim of this thesis was to evaluate the role of Regenerating Agent (RGTA) eye drops in corneal wound healing following therapeutic laser ablation of the cornea in a randomised, blinded, placebo-controlled preclinical study. Regenerating Agent is a biomimetic of extracellular matrix with well-established favourable outcomes in the treatment of skin wounds and preliminary positive results in the treatment of corneal wounds. Different manufacturing protocols were used to enhance the mechanical properties of bioengineered porcine collagen (BPC) and to address different requirements. The combination of both chemical and photochemical crosslinking with riboflavin and ultraviolet A light (to form BPCDX) and reinforcement with cellulose nanofibers extracted from the Ciona intestinalis sea invertebrate (BPCDX-CNF) resulted in stronger biomaterials compared to earlier BPC versions. The biomaterials could be manufactured in different sizes and in core-and-skirt forms with the peripheral skirt degrading faster due to mechanical compression without the addition of any cross-linkers during manufacturing. BPC could be successfully loaded with nerve growth factor (NGF) and BPCDX-CNF with dexamethasone without sacrificing transparency and both drugs could be released from BPC-based materials in vitro up to at least 2 months. The biological activity of dexamethasone released from the drug-loaded BPCDX-CNF could be confirmed by the decreased expression of inflammatory cytokines in human corneal epithelial cells grown on dexamethasone-loaded BPCDX-CNF. A compatible packaging and sterilization process was developed for BPCDX, tested internally and externally by Good Laboratory Practice-certified laboratories, that can enable worldwide distribution and storage at room temperature or in a refrigerator up to two years without the need of extra quality controls before transplantation. BPC-based materials could be safely implanted in rabbit, minipig and human corneas with advanced keratoconus using femtosecond-laser assisted intrastromal keratoplasty procedures. A femtosecond laser was used to create intrastromal pockets of different dimensions based on the size of the biomaterials. Through an access cut or by lifting a flap, both created by femtosecond laser, biomaterials could be implanted intrastromally with or without native tissue removal. Additional sutures were used in a surgical inflammatory model to test the biological activity of dexamethasone released by BPCDX-CNF. These minimally invasive surgical procedures required shorter period of immunosuppression following operation and maintained the anatomy of the surrounding host tissue. Apart from the skirt part of the composite BPC that was mechanically compressed and was designed to degrade faster, the crosslinked BPC remained stable in animal models, while no degradation was observed in the BPCDX 2 years after implantation in humans. The biomaterials were biocompatible and native cells were found in the biomaterial-host interface or in the periphery of the biomaterial. The inflammatory response following operation depended on individual response to injury and did not appear to be stimulated by BPC implantation. Neovascularization and haze formation were mainly restricted around the sutures used to secure either the access cut or flap overlying the biomaterials as well as intentionally placed close to the limbus to trigger inflammation in the dexamethasone study. In the human studies, no sutures were used and corneal transparency was maintained at the highest level in all subjects after 2 years without any signs of rejection, inflammation, vascularization or scarring. Topographic indices including mean anterior corneal curvature and maximal corneal apical curvature were significantly reduced in both clinical cohorts resulting in improved best corrected visual acuity. Importantly, following operation all human subjects could tolerate contact lenses and no subject was considered legally blind. The release of dexamethasone from the drug-loaded BPCDX-CNF could be also confirmed in vivo by sustained intraocular pressure increase, tendency to reduce neovascularization and haze formation and sustained suppression of inflammatory cytokines in the aqueous humor of eyes implanted with dexamethasone-loaded BPCDX-CNF compared to non-loaded BPCDX-CNF. Finally, RGTA eye drops following excimer laser ablation of the anterior healthy rabbit cornea did not affect the already quick and uneventful epithelial closure. Although there was significantly less haze in the RGTA group compared to placebo as measured by in vivo confocal microscopy, this decrease was not clinically relevant as all corneas in both groups were clinically transparent following laser. The microscopic haze formation and staining problems did not allow quantification of nerve regeneration, but subbasal nerves were found to repopulate the central ablated regions in both RGTA and placebo groups. In conclusion, our results indicate that biomaterials made of bioengineered porcine collagen are a safe alternative to human donor tissue for corneal transplantation, offering the advantage of custom-made manufacturing to address different requirements with potential applications to different corneal stromal diseases. Femtosecond laser enables safe intrastromal implantation of biomaterials with less suture-related issues and immunosuppression following operation compared to traditional corneal transplantation methods. Regenerating Agent eye drops do not affect the already rapid wound healing following laser ablation of the healthy cornea.
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