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Mueller, Andreas. "Assessment of eye growth-related changes in the corneal endothelium of children and young teenagers." Thesis, Glasgow Caledonian University, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.341552.
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Parekh, Mohit. "Human corneal endothelial cell culture and corneal transplantation." Doctoral thesis, Università degli studi di Padova, 2017. http://hdl.handle.net/11577/3422398.
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Cornea is the front transparent window of the eye which is responsible for optimal and clear vision. Transparency of this tissue is highly inevitable and cannot be compromised. Human cornea is made up of multiple layers out of which the posterior layer ‘endothelium’ is responsible for the transparency of the cornea. Endothelium is a monolayer of cells that allow the ions and solutes to transport from aqueous humour to the cornea and back which in turn maintains the transparency of the cornea by preserving the homeostasis between the anterior and posterior cornea. Earlier, it was observed that the endothelium had non regenerating capability however; recent studies have shown that these cells could be proliferated in vitro. Currently, the only method of treatment is the replacement of the diseased endothelium with the healthy donor endothelium. Penetrating keratoplasty which transplants a full thickness cornea was the only solution a decade ago. However, with the new advancements in the field of corneal transplants, specific surgical techniques like DMEK and DSAEK which replace only a part of the cornea have been identified. DSAEK replaces a part of the stroma along with the Descemet’s membrane and endothelium whereas DMEK only replaces the Descemet’s membrane and the endothelium and does not involve stroma. The results in terms of visual rehabilitation and outcomes have been found to be advantageous in these specific surgical procedures. However, DMEK is more challenging then DSAEK as DMEK is not yet a widespread technique, associated with steep learning curves and difficult donor tissue preparation. Despite DMEK is a challenging procedure it is becoming more popular because of the significant advantages in term of faster visual recovery, less postoperative astigmatism and reduced risk of transplant rejection, as compared to the other EK procedures. DMEK has several advantages in terms of rehabilitation rate and post-operative visual outcomes and therefore it is necessary to further refine this technique for a higher uptake of such surgeries and also considering that this is the only possible treatment for treating the patients suffering from endothelial dysfunctions. Although the corneal transplantation is well advanced, due to a limited supply of donor corneas for the transplantation purposes, alternative approaches like culturing corneal endothelium in vitro play an important role. Culturing the endothelium is not the only problem in EK but transplanting a 20 micron thick graft inside the recipient eye is another challenge. Moreover, the donor availability for culturing the corneal endothelium is less, making this strategy further more complicated. The thesis is therefore structured to highlight two significantly important issues in current scenario of endothelial keratoplasty, 1) posterior corneal transplantation or EK which is the on-going method of treatment for EK and 2) Human corneal endothelial cell culture which is the future of EK.
Chapter 1 is an introduction to the world of eye banking, its current nature and development in the modern world and as a support to the surgeons not only in terms of new techniques but also devices for selective surgeries. It also highlights the preservation of the corneal tissues which is an important element in the field of eye banking. Eye banks play a significant role in the field of corneal transplants as they collect the human corneas and process them for transplantation. The corneas that are rejected for transplantation can be used for research and therefore development of eye banking and its research can change the field of corneal transplantation. Chapter 2 introduces the field of corneal cell culture and current techniques that are followed for culturing and possible transplantation of the cultured cells.
To understand the reason and requirement of tissue engineering, it is important to study the human cornea, its extracellular matrix and its behaviour in different media. The biomechanical behaviour of the thin tissue i.e. the DM in different conditions becomes a relevant part of this study for future engineering which is studied in chapter 3. It is also important to standardize the currently available treatment options to reduce the burden of endothelial compromised patients in the future and avoid damages or tissue wastage that is currently occurring in the surgical theatres by providing standardized tissues in validated preservation medium which is studied in chapter 4. DMEK promises to become a more popular technique for the replacement of unhealthy corneal endothelium as it shows advantages like early rehabilitation rate and visual outcomes. Chapter 5 highlights the importance of new technique in rolling the DMEK tissue for easy insertion and unfolding in the recipient eye compared to the currently used technique with endothelium rolled in opposite direction. Presently, the DMEK tissues are either prepared in the surgical theatre or are stripped in the eye bank and shipped to the surgeons. However, there is no standardized procedure that could help validate a graft before surgery and provide a ready-to-use graft to the surgeons. Chapter 6 describes about a new technique of pre-loading a graft in a commercially available IOL cartridge which can be used as a preservation, transportation and transplantation device. This technique will further reduce graft wastage and will provide the surgeons a pre-validated graft further reducing the overall time in the surgical theatre and related costs. Thus different approaches for standardizing the DMEK technique were studied in the first phase of the thesis.
HCECs are currently being cultured using young donor corneas. There are two major issues, firstly, the availability of the young donor corneas is less compared to the old donor corneas and secondly, there is no standard method of culturing the HCECs obtained so far. Therefore, to reduce the global tissue demand, there is a strong need to culture the HCECs from the old donor corneas which are less proliferative and less robust in nature but with high availability of the donor source. Chapter 7 is a study on isolation of HCECs and further culture of these cells from old donor corneas. Once the protocol was obtained, a full length study was performed with high sample size to prove the consistency of this technique which is highlighted in chapter 8. Meanwhile it was also noted that cells from old donors can be cultured using ROCK inhibitor in combination with Hyaluronic Acid (HA). HA induces mechanical force to the cells attaching them forcefully on the base and allows a higher proliferation of old donor cells which was studied in chapter 9. The second part of the thesis therefore investigates the culturing technique of HCECs from old donor corneas. However, once the cells are cultured, another challenge is to transplant them in the anterior chamber of the eye. This can be performed using two strategies, first, to implant the cells as suspension in the anterior chamber which is already been proposed, but the clinical evidence is still not confirmed yet, and second, to develop a carrier to transport the cultured cells. In chapter 10, we identified fish scales as a great source of collagen and therefore have investigated it as a potential scaffold to be used for HCECs culture and transplant in the future. It is also important to understand the regulations that govern the scientific studies and its use for clinical applications. Therefore, we also identified rHSA as a source to replace FCS for preserving human corneas in chapter 11. This will also help to create a synthetic media that could be used for GMP purposes for HCECs culture in the future.
In conclusion, it was observed that pre-loading the tissues with endothelium-flapped inwards and preserved in dextran based medium could be a potential solution for providing a validated and standardized DMEK graft for the treatment of current endothelial dysfunction. Eye banks play a major role in the development of these surgical techniques and related devices which will change the face of corneal transplantation in the future. Alternatives like HCECs culture has a potential for the treatment of endothelial disorders and carriers like FSS could be used for culturing and transplanting these cells. However, the efficacy of these cells will only be validated after the clinical study. Considering the regulatory issues, synthetic medium would help both, the eye banks for preserving the corneas and its new products like pre-loaded DMEK and for cell culture in the future.<br>La cornea è quel tessuto trasparente che riveste la superficie anteriore dell'occhio, e che consente di avere una visione ottimale e chiara. La trasparenza di questo tessuto è fondamentale e non può essere compromessa. La cornea umana è costituita da più strati,tra cui lo strato posteriore o “endotelio” è responsabile della trasparenza della cornea. L’ endotelio è un monostrato di cellule che permettono agli ioni ed ai soluti di essere trasportati dall’ umor acqueo alla cornea e viceversa, e che a sua volta mantiene la trasparenza della cornea conservando l'omeostasi tra la cornea anteriore e posteriore. L’endotelio non possiede capacità rigenerative. Attualmente, l'unico metodo di trattamento è la sostituzione dell'endotelio danneggiato con l'endotelio di un donatore sano. La cheratoplastica perforante, che prevede trapianti di cornea a tutto spessore,rappresentava l'unica soluzione terapeuticafino ad un decennio fa. Tuttavia, con i nuovi progressi nel campo dei trapianti di cornea, sono state identificate specifiche tecniche chirurgiche, come DMEK e DSAEK, che sostituiscono solo una parte (o uno strato) della cornea. Sono I risultati ottenuti, in termini di riabilitazione visiva, si sono rivelati vantaggiosi grazie all’utilizzo di queste procedure chirurgiche specifiche. Tuttavia, la DMEK è più impegnativarispetto alla DSAEK in quanto non è ancora completamente standardizzata. La DMEK ha diversi vantaggi in termini di tasso di riabilitazione e risultati visivi post-operatori e quindi è necessario standardizzare questa tecnica per una maggiore diffusione di tali interventi e anche considerando che questo è l'unico trattamento possibile per la cura di pazienti affetti da disfunzioni endoteliali. Sebbene il trapianto di cornea sia in fase avanzata, a causa di una quantità limitata di cornee da donatori ai fini di trapianto, approcci alternativi come la coltura di endotelio corneale in vitro svolgono un ruolo importante. La coltura di endotelio non è l'unico problema nel trapianto di endotelio (EK)dal momento che trapiantare un innesto di 20 micron di spessore all'interno dell'occhio destinatario rappresenta una sfida ulteriore. Inoltre, la disponibilità dei donatori per la coltura di endotelio corneale è inferiore, rendendo questa strategia ulteriormente più complicata. La tesi è quindi strutturata in modo da mettere in evidenza due questioni molto importanti nell’ attuale scenario della cheratoplastica endoteliale, 1) trapianto di cornea posteriore o EK, che è l'attuale metodo di trattamento per la cheratoplastica endoteliale e 2) coltura delle cellule endoteliali della cornea umana, che rappresenta il futuro della cheratoplastica endoteliale.
Il Capitolo 1 è un'introduzione sul mondo dell’ Eye Banking, sulle sue caratteristiche attuali, sullo sviluppo nel mondo moderno e sul supporto per i chirurghi, non solo in termini di nuove tecniche, ma anche di dispositivi per interventi selettivi. Si evidenzia anche la conservazione dei tessuti corneali, che è un elemento importante nel campo dell’Eye Banking. Le banche degli occhi svolgono un ruolo significativo nel settore dei trapianti di cornea, dal momento cheraccolgono le cornee umane e le analizzano per ilsuccessivo trapianto. Le cornee non idonee per il trapianto possono essere utilizzate per la ricerca e quindi lo sviluppo dell’Eye Bankinge la ricerca possono influenzare il campo del trapianto di cornea. Il Capitolo 2 introduce l’argomento delle colture cellulari corneali e le tecniche attuali che sono utilizzate per la coltura ed il trapianto di cellule coltivate.
Per capire il motivo e l'esigenza dell’ingegnerizzazione dei tessuti, è importante studiare la cornea umana, la sua matrice extracellulare ed il suo comportamento in diversi mezzi di coltura. Il comportamento biomeccanico di un tessuto sottile (DM) in condizioni diverse rappresenta una parte rilevante di questo studio per la futura ingegnerizzazione,che viene descritta nel Capitolo 3. E’ inoltre importante standardizzare il trattamento attualmente disponibile allo scopo di ridurre in futuro l’onere di pazienti con endotelio compromesso ed evitare danni o sprechi di tessuto, che attualmente avvengono nelle sale chirurgiche, fornendo tessuti standardizzati in terreni di conservazione validati, come descritto nel Capitolo 4. La DMEK è considerata il futuro della cheratoplastica endoteliale, dal momento che presenta vantaggi quali la velocità dei tempi di riabilitazione ed i risultati visivi. Il Capitolo 5 mette in evidenza l'importanza della nuova tecnica che consiste nell’arrotolare il tessuto DMEK per consentire un facile inserimento per poi dispiegarlo nell'occhio ricevente, rispetto alla tecnica attualmente utilizzata con endotelio arrotolato in senso opposto. Attualmente, i tessuti DMEK sono o preparati in sala operatoria o allestiti in Banca degli Occhi e spediti ai chirurghi. Tuttavia, non vi è alcuna procedura standardizzata che possa contribuire ad ottenereun lembo endoteliale validato prima dell'intervento e fornire un innesto ready-to-use ai chirurghi. Il Capitolo 6 descrive una nuova tecnica di pre-caricamento di un lembo endoteliale in una cartuccia IOL disponibile in commercio che può essere utilizzato come dispositivo di conservazione, trasporto e trapianto. Questa tecnica consentirà di ridurre ulteriormente gli sprechi nei trapianti e fornirà ai chirurghi un innesto pre-convalidato,riducendo ulteriormente il tempo complessivo in sala operatoria edi relativi costi. Quindi nella prima fase della tesi, sono stati analizzati i diversi approcci per standardizzare la tecnica DMEK.
Le HCECs sono attualmente coltivate usando cornee di donatori giovani. Ci sono due aspetti importanti, in primo luogo la disponibilità di tessuti di donatori giovani è minore rispetto a quella di donatori anziani, ed in secondo luogo non vi è, ad oggi, alcun metodo standardizzato di coltura delle HCECs. Pertanto, per ridurre la domanda di tessuti a livello mondiale, vi è una forte necessità di coltivare leHCECsderivanti da cornee di donatori anziani, che sono meno proliferative e meno resistenti in natura, ma per le quali vi è una elevata disponibilità della fonte donatrice. Il Capitolo 7 descrivelo studio sull'isolamento delle HCECs e la successiva coltura di tali cellule ottenute da cornee di donatori anziani. Una volta stabilito il protocollo, è stato eseguito uno studio completocon un alto campionamento, per dimostrare la coerenza di questa tecnica,come evidenziato nel Capitolo 8. Nel frattempo si è anche osservato che le cellule da donatori anziani possono essere coltivate utilizzando l’inibitore ROCK in combinazione con acido ialuronico (HA). HA induce una forza meccanica alle cellule per far sì che siano saldamente attaccate alla base e consentire così una maggiore proliferazione,come descritto nel Capitolo 9. La seconda parte della tesi indaga quindi la tecnica di coltura delle HCECs da cornee di donatori anziani. Tuttavia, una volta che le cellule sono coltivate, un'altra sfida è trapiantarle nella camera anteriore dell'occhio. Ciò può essere eseguito utilizzando due strategie: la prima è quella di ad impiantare le cellule in forma di sospensione nella camera anteriore, tecnica che è già stata proposta, ma che non ha ancora fornito un’evidenza clinica; la secondaè quella di sviluppare un substrato per il trasporto delle cellule coltivate. Nel Capitolo 10, si identifica la colla di pesce (FSS)come una grande fonte di collagene e quindi come un potenziale scaffold da utilizzare per la cultura HCECs e successivo trapianto. E’ inoltre importante capire le norme che regolano gli studi scientifici ed il loro uso nelle applicazioni cliniche. Pertanto, nel Capitolo 11, viene descritta l’identificazione dell’ rHSA come sostitutodell’ FCS per la conservazione di cornee umane. Questo contribuirà anche a creare un terreno di coltura sintetico che potrebbe essere utilizzato per la cultura HCECs in condizioni GMP in futuro.
In conclusione, si è osservato che il pre-caricamento di tessuti con endotelio rivolto verso l'interno e conservati in un terreno con destrano, potrebbe rappresentare una possibile soluzione per fornire un lembo per DMEK validato e standardizzato per il trattamento delle disfunzioni endoteliali. Le banche degli occhi svolgono un ruolo importante nello sviluppo di queste tecniche chirurgiche e relativi dispositivi, che potranno cambiarele modalità del trapianto di cornea in futuro. Una tecnica alternativa come la coltura di HCECs ha in sèil potenziale per il trattamento di disturbi endoteliali e substrati come FSS potrebbero essere utilizzati per la coltura edil trapianto di queste cellule. Tuttavia, l'efficacia di queste cellule potrà essere validata solo dopo uno studio clinico. Considerando le questioni regolatorie, il terreno sintetico potrebbe aiutare le banche degli occhi sia per la conservazione delle cornee e dei i nuovi prodotti come DMEK pre-caricati sia, in futuro, per le colture.
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Rolev, Kostadin Dimov. "Evaluation of corneal endothelial cell therapy using an in vitro human corneal model." Thesis, Anglia Ruskin University, 2017. http://arro.anglia.ac.uk/702583/.
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Aim: To establish an in vitro human corneal decompensation model and to use it for the evaluation of a cell-therapy approach for treating corneal endothelial (CE) disorders and to test the expression profile of positive regulatory domain proteins (PRDMs) as potential markers for corneal endothelial cells (CECs). Materials and Methods: Human cadaveric corneas were obtained from Bristol and Manchester Eye Banks, UK. A CE decompensation model was established by removal of the Descemet’s membrane (DM)/Endothelium complex from donor corneas and placing them in air-interface organ culture. The corneal thickness was used as a surrogate measure of CE function and was measured using Optical Coherence Tomography (OCT). Decompensated corneas were subjected to cultured endothelial cell therapy using immortalized HCEC -12 cells (group 1), primary human corneal endothelial cells (hCECs) at 0 passage (group 2) and hCECs at passage 2 (group 3) with defined seeding cell density. The effect on stromal de-swelling in cell therapy treated corneas was assessed 3, 7 and 10 days post-transplantation followed by histological evaluation. In addition, expression of PRDM genes in the corneal endothelium was undertaken using reverse transcriptase polymerase chain reaction (RT-PCR), immunocytochemistry and immunohistochemistry. Results: Organ culture of human cadaveric corneas in air-interface following the selective removal of the DM/Endothelium complex resulted in stromal thickness of 903.6 ± 86.51 μm, whereas normal corneas maintained a physiological thickness of 557.51 ± 72.64 μm. When transplanted directly onto the posterior corneal stroma the human CECs were able to attach and achieved physiological corneal thickness of 458.91 ± 90.07 μm, 489.65 ± 94.62 μm and 613.7 ± 94.62 μm for cell therapy groups -1, -2 and -3 respectively. The study identified PRDMs 1, 2, 4, 5 and 10 in the human CE and revealed a differential expression between normal CE and cultured hCECs. Conclusion: Removal of the DM/Endothelium complex from cadaveric human corneas held in air interface organ culture resulted in corneal endothelial decompensation. Direct transplantation of cultured primary hCECs to bare posterior corneal stroma devoid of DM resulted in the formation of an endothelial monolayer and restoration of stromal hydration to physiological thickness, substantiating the role of cell therapy to treat corneal endothelial disorders. The identification of PRDM proteins in the human corneal endothelium paves the way for future studies to understand their role in hCEC proliferation control.
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Bethi, Akhila. "Endothelin-1 Induced Phosphorylation of ERK1/2 in Bovine Corneal Endothelial Cells." TopSCHOLAR®, 2012. http://digitalcommons.wku.edu/theses/1191.
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The purpose of this study was to determine whether Endothelin-1 (ET-1) induced cellular responses in bovine corneal endothelial cells (BCECs) involves MAPK pathway by phosphorylating ERK1/2 protein kinase and to find out the phosphorylation patterns of ERK1/2 in confluent and sub-confluent cells. BCECs were isolated from bovine corneas and cultured in medium supplemented with 10% serum. Confluent (contact inhibited) and sub-confluent (actively growing cells) serum starved cells grown in T-75 flasks were treated with 10nM Endothelin-1. The control cells were left untreated. Total cellular protein was isolated using RIPA buffer and quantified according to the Peterson modification of the Lowry method. The level of expression of phosphorylated ERK1/2 (pp44, pp42) proteins relative to overall ERK1/2 (p44, p42) was determined by western blotting technique. Densitometry analysis of immunoblots revealed differential phosphorylation patterns in confluent and sub-confluent cultures. The pERK1/2 levels were significantly increased at 15 min and 24 hrs after post incubation with ET-1, whereas following the initial rise levels declined to 6hrs of incubation with ET-1 in confluent cultures. In sub-confluent cultures pERK1/2 levels increased gradually to 6hrs of incubation with ET-1, returning to pre-incubation levels at 24hrs. In conclusion, ET-1 treatment was shown to induce phosphorylation of ERK1/2 in BCEC. ET-1 treatment in confluent and sub confluent BCEC exhibited time dependent phosphorylation of ERK1/2. ET-1 treatment affected the phosphorylation pattern distinctively in confluent and sub-confluent BCEC. These observations led to the conclusion that ET-1 induced cellular events in BCEC may involve the MAPK cascade and that these ET-1 induced MAPK cascades may exhibit a negative feedback mechanism, suggested by a distinctive oscillations in pERK 1/2 levels. The contrasting effects of ET-1 in confluent and subconfluent cells may suggest a density dependent phosphatase activity.
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Jackson, Andrew Jonathan. "An analysis of corneal endothelial cell morphology under normal and traumatic conditions." Thesis, Queen's University Belfast, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.333818.
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Al, Abdulsalam Najla Khaled S. "Evaluation of silk fibroin as a scaffold for cultured corneal endothelial cell implants." Thesis, Queensland University of Technology, 2018. https://eprints.qut.edu.au/122231/1/Najla%20Khaled%20S_Al%20Abdulsalam_Thesis.pdf.
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Corneal transplants are a safe and effective treatment for corneal disease, but are hampered by the limited supply and quality of donor tissue. The goal of this project was therefore to evaluate the use of membranes prepared from silk protein as a scaffold on which to grow corneal tissue substitutes in the laboratory from corneal endothelial cells.
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Valtink, Monika, Rita Gruschwitz, Richard H. W. Funk, and Katrin Engelmann. "Two Clonal Cell Lines of Immortalized Human Corneal Endothelial Cells Show either Differentiated or Precursor Cell Characteristics." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-136199.
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Access to primary human corneal endothelial cells (HCEC) is limited and donor-derived differences between cultures exacerbate the issue of data reproducibility, whereas cell lines can provide sufficient numbers of homogenous cells for multiple experiments. An immortalized HCEC population was adapted to serum-free culture medium and repeated cloning was performed. Clonally grown cells were propagated under serum-free conditions and growth curves were recorded. Cells were characterized immunocytochemically for junctional proteins, collagens, Na,K-ATPase and HCEC-specific 9.3.E-antigen. Ultrastructure was monitored by scanning and transmission electron microscopy. Two clonal cell lines, HCEC-B4G12 and HCEC-H9C1, could be isolated and expanded, which differed morphologically: B4G12 cells were polygonal, strongly adherent and formed a strict monolayer, H9C1 cells were less adherent and formed floating spheres. The generation time of B4G12 cells was 62.26 ± 14.5 h and that of H9C1 cells 44.05 ± 5.05 h. Scanning electron microscopy revealed that B4G12 cells had a smooth cell surface, while H9C1 cells had numerous thin filopodia. Both cell lines expressed ZO-1 and occludin adequately, and little but well detectable amounts of connexin-43. Expression of HCEC-specific 9.3.E-antigen was found commensurately in both cell lines, while expression of Na,K-ATPase α1 was higher in H9C1 cells than in B4G12 cells. B4G12 cells expressed collagen IV abundantly and almost no collagen III, while H9C1 cells expressed both collagens at reasonable amounts. It is concluded that the clonal cell line B4G12 represents an ideal model of differentiated HCEC, while H9C1 may reflect features of developing or transitional HCEC<br>Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich
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Valtink, Monika, Rita Gruschwitz, Richard H. W. Funk, and Katrin Engelmann. "Two Clonal Cell Lines of Immortalized Human Corneal Endothelial Cells Show either Differentiated or Precursor Cell Characteristics." Karger, 2008. https://tud.qucosa.de/id/qucosa%3A27701.
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Access to primary human corneal endothelial cells (HCEC) is limited and donor-derived differences between cultures exacerbate the issue of data reproducibility, whereas cell lines can provide sufficient numbers of homogenous cells for multiple experiments. An immortalized HCEC population was adapted to serum-free culture medium and repeated cloning was performed. Clonally grown cells were propagated under serum-free conditions and growth curves were recorded. Cells were characterized immunocytochemically for junctional proteins, collagens, Na,K-ATPase and HCEC-specific 9.3.E-antigen. Ultrastructure was monitored by scanning and transmission electron microscopy. Two clonal cell lines, HCEC-B4G12 and HCEC-H9C1, could be isolated and expanded, which differed morphologically: B4G12 cells were polygonal, strongly adherent and formed a strict monolayer, H9C1 cells were less adherent and formed floating spheres. The generation time of B4G12 cells was 62.26 ± 14.5 h and that of H9C1 cells 44.05 ± 5.05 h. Scanning electron microscopy revealed that B4G12 cells had a smooth cell surface, while H9C1 cells had numerous thin filopodia. Both cell lines expressed ZO-1 and occludin adequately, and little but well detectable amounts of connexin-43. Expression of HCEC-specific 9.3.E-antigen was found commensurately in both cell lines, while expression of Na,K-ATPase α1 was higher in H9C1 cells than in B4G12 cells. B4G12 cells expressed collagen IV abundantly and almost no collagen III, while H9C1 cells expressed both collagens at reasonable amounts. It is concluded that the clonal cell line B4G12 represents an ideal model of differentiated HCEC, while H9C1 may reflect features of developing or transitional HCEC.<br>Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.
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Kocaba, Viridiana. "Tissue engineering pour la reconstruction cornéenne." Thesis, Lyon, 2018. http://www.theses.fr/2018LYSE1078.
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En France, les dysfonctions endothéliales représentent environ la moitié des indications de greffes de cornée réalisées chaque année. Cependant, les problématiques liées à la pénurie de greffon, aux difficultés des techniques chirurgicales de greffes endothéliales ainsi qu’aux risques d’échec ou de rejet de greffe poussent les chercheurs à développer de nouvelles thérapies moins invasives et plus efficaces. La thérapie cellulaire cornéenne endothéliale est une des voies de recherche actuellement explorées dont le but est de s’affranchir des aléas de la greffe de cornée. La cornée humaine est un tissu idéal pour la thérapie cellulaire. Grâce à ses caractéristiques d’organe à la fois avasculaire et immunitairement privilégié, les cellules transplantées sont ainsi bien mieux tolérées par rapport aux autres tissus et organes vascularisés. Les avancées dans le domaine des cellules souches, de l'ingénierie, particulièrement avec l’arrivée des greffes de cellules souches épithéliales pour le traitement des pathologies sévères de la surface oculaire, ont suscité un intérêt massif afin d’adapter ces techniques aux cellules endothéliales<br>In France, around half of all corneal keratoplasties are performed to treat corneal endothelial dysfunction each year. However, the use of endothelial keratoplasty is limited by the technical difficulty of the procedure, a shortage of available grafts, and the potential for graft failure or rejection. These limitations are driving researchers to develop new, less invasive, and more effective therapies. Corneal endothelial cell therapy is being explored as a potential therapeutic measure, to avoid the uncertainty associated with grafting. The human cornea is an ideal tissue for cell therapy as owing to its avascular characteristics, transplanted cells are better tolerated compared with other vascularized tissues and organs. Advances in the field of stem-cell engineering, particularly the development of corneal epithelial stem cell therapy for the treatment of severe diseases of the ocular surface, have aroused a massive interest in adapting cell-therapy techniques to corneal endothelial cells
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Bollu, Lakshmi Reddy. "The Effect of Endothelin-1 on the expression of CDK Inhibitors p21 & p27 in Bovine Corneal Endothelial Cells." TopSCHOLAR®, 2009. http://digitalcommons.wku.edu/theses/112.
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Mammalian corneal endothelial cells are considered to be non-proliferative due to the arrest of cells at the G1 phase of the cell cycle. The purpose of this study was to determine whether the down regulation of cyclin dependant kinase inhibitors (p21cip1 and p27kip1) levels by Endothelin-1 (ET-1), would overcome the G1 phase arrest and promote cell cycle progression and proliferation in cultured BCECs (Bovine corneal endothelial cells). BCECs were isolated from bovine corneas and cultured in DMEM supplemented with 10% serum. 5-Bromo 2-deoxy Uridine (BrdU) incorporation was determined in serum starved cultures in 24-well plates as a measure of cell proliferation. Confluent serum starved cells grown in T-25 flasks were treated with 100nM Endothelin-1 in DMEM. The control cells were left untreated in serum free medium. Total cellular protein was isolated using RIPA buffer and quantified according to the Peterson modification of the Lowry method. The level of expression of p21cip1 and p27kip1 proteins relative to β-actin was determined by western blotting technique. Immuno fluorescent localization of p27kip1 was performed using polyclonal anti-p27kip1 and anti-p21cip1 antibodies in confluent and growing cells. An increase in cell proliferation was observed in sub-confluent cultures with Endothelin-1 treatment. This evidence was supported by an increase (~18%) in BrdU incorporation in response to Endothelin-1. Densitometry analysis of immunoblots revealed an increase in the expression of p27kip1 in confluent cell cultures when compared to sub-confluent, dividing cells. p21cip1 was almost undetectable in sub-confluent, actively dividing cultures. Immuno fluorescent analysis revealed that the nuclear staining of p27kip1 was apparently decreased with ET-1 treatment. In conclusion, Endothelin-1 treatment resulted in decrease in p27kip1 and p21cip1 expression in confluent cultures that was greatest at 30 hr of post incubation with Endothelin-1. Endothelin-1 appears to promote cell proliferation. Expression of p27kip1 and p21cip1 was greatly reduced in actively dividing BCECs. Endothelin-1 treatment down-regulated these cyclin dependent kinase inhibitors and may promote cell cycle progression via this mechanism.
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Xie, Qiang. "Studies on calcium(2+) signaling in corneal endothelial cells and molecular characterization of a novel apoptosis-inducing factor like gene." [Bloomington, Ind.] : Indiana University, 2004. http://wwwlib.umi.com/dissertations/fullcit/3162272.
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Thesis (Ph.D.)--Indiana University, School of Optometry, 2004.<br>Title from PDF t.p. (viewed Dec. 2, 2008). Source: Dissertation Abstracts International, Volume: 66-01, Section: B, page: 0050. Chair: Joseph A. Bonanno.
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Lutz, Elizabeth Anne. "Effects of Modified Cyclosporine A on Posterior Capsule Opacification Formation and Corneal Endothelial Cell Viability in an Ex Vivo Model." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1371477702.
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Huang, Lan. "Endothelial Colony Forming Cells (ECFCs): Identification, Specification and Modulation in Cardiovascular Diseases." Thesis, Connect to resource online, 2009. http://hdl.handle.net/1805/2063.
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Thesis (Ph.D.)--Indiana University, 2009.<br>Title from screen (viewed on February 2, 2010). Department of Biochemistry and Molecular Biology, Indiana University-Purdue University Indianapolis (IUPUI). Advisor(s): Mervin C. Yoder, Jr., David A. Ingram, Jr., Lawrence A. Quilliam, Mark D. Pescovitz. Includes vitae. Includes bibliographical references (leaves 171-194).
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Kuzuya, Fumio, Kanichi Asai, Toshio Hayashi, Chiaki Funaki, Michitaka Naito, and Masafumi Kuzuya. "Lipid peroxide and transition metals are required for the toxicity of oxidized low density lipoprotein to cultured endothelial cells." Thesis, Elsevier, 1991. http://hdl.handle.net/2237/16697.
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Català, Mora Jaume. "Implante de lente de anclaje iridiano para el tratamiento de la ectopia lentis no traumática. Seguimiento a largo plazo en una cohorte de pacientes pediátricos." Doctoral thesis, Universitat Autònoma de Barcelona, 2016. http://hdl.handle.net/10803/399779.
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La ectopia lentis (EL) consiste en un desplazamiento del cristalino idiopático o asociado a diferentes alteraciones sistémicas o metabólicas, incluyendo el síndrome de Marfan, homocistinuria y el síndrome de Weill-Marchesiani. La EL también puede ocurrir en casos de megaloftalmos anterior idiopático o secundario. En estos pacientes se produce una pérdida visual secundaria a la subluxación del cristalino, que puede provocar alta miopía y astigmatismo. Además, el borde del cristalino subluxado podría interferir con el eje visual, induciendo alteraciones visuales que podrían provocar ambliopía. Otras complicaciones potenciales de la EL incluyen luxación del cristalino en el vítreo o en la cámara anterior, donde podría lesionar el endotelio corneal o provocar un bloqueo pupilar con hipertensión ocular por cierre angular.
El manejo de la EL congénita progresiva es controvertido. Se han propuesto múltiples indicaciones y técnicas quirúrgicas. La extracción del cristalino se puede realizar mediante facoaspiración como a través de lensectomía vía pars plana y vitrectomía. Una vez se ha retirado el cristalino, debemos corregir la afaquia mediante gafas, lentes de contacto o con el implante de una lente intraocular (LIO). Existen múltiples tipos de LIO: LIO de cámara posterior con fijación escleral, anillos de Cionni de fijación escleral y LIO sacular, LIO de cámara anterior y LIO de fijación iridiana pre o retropupilar. La elección del tipo de implante en pacientes pediátricos es una decisión compleja, aunque la mayor parte de cirujanos prefieren LIO de fijación iridiana o escleral en este grupo de pacientes.
Hemos llevado a cabo un estudio prospectivo en una cohorte de 21 ojos de 12 pacientes con ectopia lentis severa y AV inferior a 20/63. Se practicó una vitrectomía 23 G vía pars plana, lensectomía, iridectomía e implante de LIO Artisan en la cámara anterior con un enclavamiento vía pars plana. La edad media en el momento de la cirugía fue de 8,0 ± 5.3 años (rango 3-17 años). Se realizó una exploración oftalmológica completa con mejor agudeza visual corregida (MAVC), biomicroscopía, toma de la presión intraocular, estudio de fondo de ojo y recuento endotelial corneal central (DCEC) antes del tratamiento, a los 3 meses de la intervención y cada 6 meses. A los 12 meses de la cirugía se realizó una biomicroscopía ultrasónica.
El seguimiento medio fue de 39,3 ± 13,0 meses. La MAVC (media ± desviación típica) mejoró desde 0,91 ± 0,29 LogMar en el preoperatorio hasta 0,18 ± 0,23 LogMar al final del seguimiento (p < 0.0001). La distancia media entre el endotelio y la superficie anterior de la LIO después de la cirugía fue de 3,11 ± 0,61 mm. La pérdida endotelial en el postoperatorio inmediato fue del 5,04% ± 9,58% con una perdida endotelial anual del 3,16% ± 4,46%. Un paciente sufrió una luxación de la LIO y un desprendimiento de retina tras una contusión ocular severa y fue tratado con vitrectomía, recolocación de LIO y tamponamiento con gas. Otro paciente desarrolló un edema macular quístico y fue tratado con dexametasona intravítrea.
Conclusiones:
La lensectomía vía pars plana con implante de LIO preiridiana con fijación iridiana por vía posterior mejora la visión de manera significativa en niños con ectopia lentis no traumática.
La pérdida endotelial en el postoperatorio inmediato supone un 5.04 ± 9.58 %., seguida de una pérdida endotelial anual del 3.16 ± 4.56 %. Estos valores son similares a los descritos con otras técnicas y no son superiores a los valores esperados en la población pediátrica.
No se ha encontrado relación directa entre la distancia endotelio-LIO y la pérdida endotelial. La pérdida de un 25% de la población corneal endotelial se asocia, sin alcanzar significación estadística, con una menor distancia endotelio-LIO.
Se han observado menos complicaciones que con otras técnicas de enclavamiento de la LIO de fijación iridiana.<br>Ectopia lentis (EL) is a displacement of the crystalline lens that can be idiopathic or in the setting of various systemic and metabolic disorders, including Marfan's syndrome, homocystinuria, and Weill-Marchesani syndrome. EL can also occur in cases of idiopathic or secondary anterior megalophthalmos. Visual impairment in these patients is secondary to crystalline lens subluxation which may cause high myopia and astigmatism. In addition, the border of the subluxated lens can interfere with the visual axis, thus inducing visual disturbances that could lead to amblyopia. Potential complications of ectopia lentis include dislocation of the lens into the vitreous or anterior chamber, where it can damage the corneal endothelium or induce a pupillary block and angle closure ocular hypertension.
The management of progressive congenital EL is controversial. A wide variety of indications and lens removal techniques have been proposed. Both phacoaspiration or pars plana lensectomy and vitrectomy can be used for lens extraction. Once the lens is removed, aphakia must be corrected with glasses, contact lens or intraocular lens (IOL) implantation. A variety of IOL types are available, including scleral–fixated posterior chamber IOL, scleral-fixated Cionni ring, in-the-bag IOL, anterior chamber IOL, and anterior or posteriorly implanted iris-claw IOL. The choice of the IOL implant in paediatric patients is highly challenging, although most surgeons prefer iris or scleral-sutured IOL in this patient population.
We conducted a prospective cohort study of 21 eyes from 12 patients with severe ectopia lentis and visual acuity < 20/63. All eyes underwent 23-gauge pars plana vitrectomy, lensectomy, iridectomy, and Artisan IOL implantation in the anterior chamber with iris-claw enclavation via pars plana. Mean age at surgery was 8.0 ± 5.3 y.o. (range 3-17 years). A full ophthalmologic examination including best corrected visual acuity (BCVA), biomicroscopy, intraocular pressure (IOP) measurement, fundus evaluation, and central endothelial cell count (cECC) was performed pre-treatment, at 3 months’ post-surgery, and every 6 months thereafter. Ultrasound biomicroscopy was performed 12 months after surgery.
Mean follow-up was 39.3 ± 13.0 months. BCVA (mean ± standard deviation) improved from 0.91 ± 0.29 LogMar preoperatively to 0.18 ± 0.23 LogMar at final follow-up (p < 0.0001). Mean distance from the endothelium to the anterior IOL surface after surgery was 3.11 ± 0.61 mm. Postsurgically, cECC loss was 5.04% ± 9.58% with an annual cECC loss rate of 3.16% ± 4.46%. One patient developed IOL dislocation and retinal detachment after severe ocular contusion requiring vitrectomy, IOL refixation, and gas tamponade. Another patient developed cystoid macular oedema, managed with intravitreal dexamethasone.
Conclusion:
Pars plana lensectomy and anterior iris-claw implant with posterior fixation improves significantly visual acuity in children with non-traumatic severe ectopia lentis.
Endothelial cell loss after surgery achieves a 5,04 ± 9,58 % followed by an annual endothelial cell loss of 3,16 ± 4,56 %. These data are similar to other techniques and they are not higher of what should be expected in the paediatric population.
It hasn’t been found a direct relationship between the distance between central corneal endothelium and anterior IOL surface The loss of 25% of the central corneal endothelial population is related, without reaching a statistical significance, to lower endothelium-IOL distances.
We have found a lower rate of complications compared to other iris IOL fixation techniques.
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Hammadi, Shumoos T. H. "Novel medical imaging technologies for processing epithelium and endothelium layers in corneal confocal images. Developing automated segmentation and quantification algorithms for processing sub-basal epithelium nerves and endothelial cells for early diagnosis of diabetic neuropathy in corneal confocal microscope images." Thesis, University of Bradford, 2018. http://hdl.handle.net/10454/16924.
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Diabetic Peripheral Neuropathy (DPN) is one of the most common types of diabetes that can affect the cornea. An accurate analysis of the corneal epithelium nerve structures and the corneal endothelial cell can assist early diagnosis of this disease and other corneal diseases, which can lead to visual impairment and then to blindness. In this thesis, fully-automated segmentation and quantification algorithms for processing and analysing sub-basal epithelium nerves and endothelial cells are proposed for early diagnosis of diabetic neuropathy in Corneal Confocal Microscopy (CCM) images. Firstly, a fully automatic nerve segmentation system for corneal confocal microscope images is proposed. The performance of the proposed system is evaluated against manually traced images with an execution time of the prototype is 13 seconds. Secondly, an automatic corneal nerve registration system is proposed. The main aim of this system is to produce a new informative corneal image that contains structural and functional information. Thirdly, an automated real-time system, termed the Corneal Endothelium Analysis System (CEAS) is developed and applied for the segmentation of endothelial cells in images of human cornea obtained by In Vivo CCM. The performance of the proposed CEAS system was tested against manually traced images with an execution time of only 6 seconds per image. Finally, the results obtained from all the proposed approaches have been evaluated and validated by an expert advisory board from two institutes, they are the Division of Medicine, Weill Cornell Medicine-Qatar, Doha, Qatar and the Manchester Royal Eye Hospital, Centre for Endocrinology and Diabetes, UK.
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Daher, Jalil. "Exposure of endothelial cells to physiological levels of myeloperoxidase modified LDL delays pericellular fibrinolysis and reduces cell motility." Doctoral thesis, Universite Libre de Bruxelles, 2014. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/209337.
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Cardiovascular diseases are considered the first cause of death in westernized societies. They are directly linked to atherosclerosis, a clinical condition characterized by a thickening of the arterial wall. Atherosclerosis is in his turn linked to various genetic and environmental factors; among those factors are high oxidized LDL levels and endothelial dysfunction. In the present study, we have analyzed in vitro the effect of myeloperoxidase oxidized LDL on endothelial cells at the level of fibrinolysis and cell motility.<p>In the first part of the work, we measured fibrinolysis in real time at the surface of endothelial cells. Our results suggest that myeloperoxidase oxidized LDL interferes with the regulation of fibrinolysis by endothelial cells by decreasing their pro-fibrinolytic activity. This effect was not related to a modification in expression of major regulators of fibrinolysis such as PAI-1 and t-PA. Our data link the current favorite hypothesis that oxidized LDL has a causal role in atheroma plaque formation with an old suggestion that fibrin may also play a causal role. A model that best explains our results would be as follows: oxidized LDL increases fibrin deposition on endothelial cells which will increase their permeability resulting in more oxidized LDL infiltration into the subendothelial space of the arterial wall initiating atherogenesis. <p>In the second part of the work, we investigated the effect of myeloperoxidase oxidized LDL at the level of endothelial cell motility. We have shown that oxidized LDL is able to decrease cell migration, wound healing and tubulogenesis in endothelial cells. Those effects were not associated with any alteration at the level of neither cell viability nor proliferation. Subsequent gene expression analyses enabled us to link the oxidized LDL induced phenotypical changes in the cells to a change in expression of both microRNA-22 and Heme Oxygenase 1 genes. Our observations suggest a novel role of oxidized LDL not only as an important factor in the initiation of atheromatous lesions, but also as a potential player in the progression of the atherosclerosis disease by impeding blood vessel repair and wound healing at the sites of lesions.<p><br>Doctorat en Sciences<br>info:eu-repo/semantics/nonPublished
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Cui, Yuqi. "Role of Reactive Oxygen Species in Mediating the Effect of Oxidized Low Density Lipoprotein on Bone Marrow Stem Cells and Endothelial Progenitor Cells in Hyperlipidemia." The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1397058562.
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He, Zhiguo. "Application de l'immunolocalisation à la recherche de la cellule souche endothéliale cornéenne humaine." Phd thesis, Université Jean Monnet - Saint-Etienne, 2011. http://tel.archives-ouvertes.fr/tel-00987968.
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Le contrôle de la transparence de la cornée dépend de l'intégrité de l'endothélium cornéen mono-stratifié qui est classiquement considéré dès la naissance, dépourvu de capacité de régénération chez l'homme. Dans des conditions pathologiques conduisant à la cécité par œdème cornéen, les pertes significatives en cellules endothéliales (CE) ne sont pas remplacée efficacement, ce qui signifie que ni de nouvelles CE provenant de cellules souches (CS), ni la division des cellules voisines des lésions ne peuvent contribuer à la régénération endothéliale. Toutefois, plusieurs travaux ont prouvé depuis 25 ans que les CE possédaient une capacité proliférative résiduelle ex vivo et deux équipes ont suggéré l'existence de CS ou de progéniteurs à la périphérie de l'endothélium cornéen. Dans notre travail de thèse, nous avons tout d'abord optimisé, en la systématisant, une technique d'immunomarquage spécialement adaptée à l'endothélium cornéen intact de cornées montées à plat. A l'issue de ces développements, nous disposons de protocoles simples de fixation à température optimale et de démasquages antigéniques susceptibles de permettre la révélation de nombreuses protéines. A partir d'une importante série de cornées humaines non conservées et d'autres conservées en organoculture, et grâce à cet outil désormais efficace, nous avons étudié le cycle cellulaire des CE et la localisation de potentielles CS sur l'endothélium cornéen humaine. Nos résultats démontrent que dans ces conditions, les CE expriment de façon homogène des régulateurs positifs (PCNA, MCM2, cycline D1, cycline E et cycline A) et des régulateurs négatifs du cycle cellulaire (P16, P27); certaines particularités ont par ailleurs pu être décrites de façon innovante, comme la localisation cytoplasmique diffuse de MCM2, paranucléaire de la cycline D1, l'absence de P21. L'ensemble des marquages pourrait suggérer que les CE sont arrêtées en fin de G1, après le point de restriction et que de nombreux mécanismes de réparation de l'ADN sont mis en jeux dans les CE exposées à un stress oxydant important tout au long de l'existence. Nous avons identifié une nouvelle organisation de la micro-anatomie de la périphérie et de l'extrême périphérie de l'endothélium où des cellules regroupées en multiples clusters pluristratifiés semblent alimenter des colonnes de CE radiaires longues d'un millimètre. Ces éléments, associés à l'observation d'une moindre différenciation et d'une compétence proliférative plus élevés en périphérie suggèrent un nouveau modèle d'homéostasie endothéliale humaine in vivo: toute la vie, des CS périphériques alimentent de façon très lente la périphérie cornéenne en CE qui migrent de façon centripète pour assurer la stabilité du centre cornéen dont les propriétés optiques primordiales sont sous-tendues par un endothélium qui ne perd que 0,6% de CE par an. A la différence de l'épithélium cornéen, ce système ne peut être accéléré lors de circonstances pathologiques. Les perspectives de nos travaux sont désormais d'essayer d'isoler de l'extrême périphérie les CS endothéliales ou les progéniteurs et de les cultiver en recréant un microenvironnement favorable. La possibilité de produire un grand nombre de CE in vitre non pas à partir de CE sénescentes prélevées sur la totalité de l'endothélium comme cela a été tenté par la passé, mais cette fois à partir de CS ou des progéniteurs ouvriraient la voie d'une véritable thérapie cellulaire endothéliale. L'enrichissement des greffons pendant la durée de leur conservation à la banque de cornée pourrait constituer une première étape majeure avant d'envisager créer de novo un endothélium sur un support greffable pour une greffe endothéliale du 3e type qui deviendrait ainsi enfin indépendante des aléas de la découpe du greffon. Enfin, l'ïdentification de la CS endothéliale et de son microenvironnement permettra aussi d'envisager une thérapie cellulaire in vivo pour traiter les stades précoces des pathologies endothéliales cornéennes
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Rolev, Kostadin D. "Evaluation of corneal endothelial cell therapy using an in vitro human corneal model." Thesis, 2017. https://arro.anglia.ac.uk/id/eprint/702583/1/Rolev_2017.pdf.
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Aim: To establish an in vitro human corneal decompensation model and to use it for the evaluation of a cell-therapy approach for treating corneal endothelial (CE) disorders and to test the expression profile of positive regulatory domain proteins (PRDMs) as potential markers for corneal endothelial cells (CECs).
Materials and Methods: Human cadaveric corneas were obtained from Bristol and Manchester Eye Banks, UK. A CE decompensation model was established by removal of the Descemet’s membrane (DM)/Endothelium complex from donor corneas and placing them in air-interface organ culture. The corneal thickness was used as a surrogate measure of CE function and was measured using Optical Coherence Tomography (OCT). Decompensated corneas were subjected to cultured endothelial cell therapy using immortalized HCEC -12 cells (group 1), primary human corneal endothelial cells (hCECs) at 0 passage (group 2) and hCECs at passage 2 (group 3) with defined seeding cell density. The effect on stromal de-swelling in cell therapy treated corneas was assessed 3, 7 and 10 days post-transplantation followed by histological evaluation. In addition, expression of PRDM genes in the corneal endothelium was undertaken using reverse transcriptase polymerase chain reaction (RT-PCR), immunocytochemistry and immunohistochemistry.
Results: Organ culture of human cadaveric corneas in air-interface following the selective removal of the DM/Endothelium complex resulted in stromal thickness of 903.6 ± 86.51 μm, whereas normal corneas maintained a physiological thickness of 557.51 ± 72.64 μm. When transplanted directly onto the posterior corneal stroma the human CECs were able to attach and achieved physiological corneal thickness of 458.91 ± 90.07 μm, 489.65 ± 94.62 μm and 613.7 ± 94.62 μm for cell therapy groups -1, -2 and -3 respectively. The study identified PRDMs 1, 2, 4, 5 and 10 in the human CE and revealed a differential expression between normal CE and cultured hCECs.
Conclusion: Removal of the DM/Endothelium complex from cadaveric human corneas held in air interface organ culture resulted in corneal endothelial decompensation. Direct transplantation of cultured primary hCECs to bare posterior corneal stroma devoid of DM resulted in the formation of an endothelial monolayer and restoration of stromal hydration to physiological thickness, substantiating the role of cell therapy to treat corneal endothelial disorders. The identification of PRDM proteins in the human corneal endothelium paves the way for future studies to understand their role in hCEC proliferation control.
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Lo, Hung-Fei, and 駱虹霏. "Phosphotyrosine Signaling as a Regulator of Corneal Endothelial Cell Function." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/57418862968572002520.
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碩士<br>國立臺灣大學<br>獸醫學研究所<br>93<br>The importance of phosphotyrosine signal transduction in cellular proliferation, cell-cell contact and cellular migration has been proved in various cell types. However, only limited studies have been reported on corneal endothelial cell cells. In this study, we aim to understand the role of phosphotyrosine signaling in corneal endothelial cellular function. We propose that protein tyrosine phosphotase (PTP) may play an important role in cell-cell junction of corneal endothelial cells, and the disruption of its function by PTP inhibitor can break through cell-cell junction, and trigger a lot of downstream actions such as cellular proliferation and change of permeability. Primary culture of bovine corneal endothelial cells and whole rabbit corneas were used as the experimental materials. We first treated the cultured bovine corneal endothelial cells with PTPs inhibitor, sodium orthovanadate (SOV), with a variety of concentrations (25,50,100μM) for various durations (8,24 hrs). The effects of PTP inhibition on cellular distribution of cell-cell junctional proteins, such as N-cadherin, alpha-catenin and p120, were evaluated by immunohistochemical staining with fluorescein microscopy and confocal microscopy. Immunohistochemical staining with Ki67 Ab, a marker of cell proliferation, was also used to detect cells entering cell cycle. The expression levels of these cellular junctional proteins and regulatory proteins in cell cycle regulation such as Cyclin A, Cyclin E, Cyclin D1 and PCNA, were quantified by Western blotting. Our results demonstrate that PTPs inhibitor broke through cell-cell junction, and triggered corneal endothelial cells to re-enter cell cycle instead of having no impacts on celluar proliferation. However, the expression levels of those chosen cell-cell junction proteins and chosen regulatory proteins in cell cycle regulation were unchanged. Further studies will be carried out to elucidate the mechanism of phosphotyrosine signaling in mediating the cellular behavior of corneal endothelial cells.
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Al-Fahdawi, Shumoos, Rami S. R. Qahwaji, Alaa S. Al-Waisy, et al. "A fully automated cell segmentation and morphometric parameter system for quantifying corneal endothelial cell morphology." 2018. http://hdl.handle.net/10454/15684.
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Yes<br>Background and Objective
Corneal endothelial cell abnormalities may be associated with a number of corneal and systemic diseases. Damage to the endothelial cells can significantly affect corneal transparency by altering hydration of the corneal stroma, which can lead to irreversible endothelial cell pathology requiring corneal transplantation. To date, quantitative analysis of endothelial cell abnormalities has been manually performed by ophthalmologists using time consuming and highly subjective semi-automatic tools, which require an operator interaction. We developed and applied a fully-automated and real-time system, termed the Corneal Endothelium Analysis System (CEAS) for the segmentation and computation of endothelial cells in images of the human cornea obtained by in vivo corneal confocal microscopy.
Methods
First, a Fast Fourier Transform (FFT) Band-pass filter is applied to reduce noise and enhance the image quality to make the cells more visible. Secondly, endothelial cell boundaries are detected using watershed transformations and Voronoi tessellations to accurately quantify the morphological parameters of the human corneal endothelial cells. The performance of the automated segmentation system was tested against manually traced ground-truth images based on a database consisting of 40 corneal confocal endothelial cell images in terms of segmentation accuracy and obtained clinical features. In addition, the robustness and efficiency of the proposed CEAS system were compared with manually obtained cell densities using a separate database of 40 images from controls (n = 11), obese subjects (n = 16) and patients with diabetes (n = 13).
Results
The Pearson correlation coefficient between automated and manual endothelial cell densities is 0.9 (p < 0.0001) and a Bland–Altman plot shows that 95% of the data are between the 2SD agreement lines.
Conclusions
We demonstrate the effectiveness and robustness of the CEAS system, and the possibility of utilizing it in a real world clinical setting to enable rapid diagnosis and for patient follow-up, with an execution time of only 6 seconds per image.
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Haydari, M. Nour. "Regenerative potential of corneal endothelium from patients with fuchs endothelial corneal dystrophy." Thèse, 2012. http://hdl.handle.net/1866/10115.
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La dystrophie cornéenne endothéliale de Fuchs (FECD, pour l’abréviation du terme anglais « Fuchs endothelial corneal dystrophy ») est une maladie de l'endothélium cornéen. Sa pathogenèse est mal connue. Aucun traitement médical n’est efficace. Le seul traitement existant est chirurgical et consiste dans le remplacement de l’endothélium pathologique par un endothélium sain provenant de cornées de la Banque des yeux. Le traitement chirurgical, en revanche, comporte 10% de rejet immunologique. Des modèles expérimentaux sont donc nécessaires afin de mieux comprendre cette maladie ainsi que pour le développement de traitements alternatifs. Le but général de cette thèse est de développer un modèle expérimental de la FECD en utilisant le génie tissulaire. Ceci a été réalisé en trois étapes. 1) Tout d'abord, l'endothélium cornéen a été reconstruit par génie tissulaire en utilisant des cellules endothéliales en culture, provenant de patients atteints de FECD. Ce modèle a ensuite été caractérisé in vitro. Brièvement, les cellules endothéliales cornéennes FECD ont été isolées à partir de membranes de Descemet prélevées lors de greffes de cornée. Les cellules au deuxième ou troisième passages ont ensuite été ensemencées sur une cornée humaine préalablement décellularisée. Suivant 2 semaines de culture, les endothélia cornéens reconstruits FECD (n = 6) ont été évalués à l'aide d'histologie, de microscopie électronique à transmission et d’immunomarquages de différentes protéines. Les endothélia cornéens reconstruits FECD ont formé une monocouche de cellules polygonales bien adhérées à la membrane de Descemet. Les immunomarquages ont démontré la présence des protéines importantes pour la fonctionnalité de l’endothélium cornéen telles que Na+-K+/ATPase α1 et Na+/HCO3-, ainsi qu’une expression faible et uniforme de la protéine clusterine. 2) Deux techniques chirurgicales (DSAEK ; pour « Descemet stripping automated endothelial keratoplasty » et la kératoplastie pénétrante) ont été comparées pour la transplantation cornéenne dans le modèle animal félin. Les paramètres comparés incluaient les défis chirurgicaux et les résultats cliniques. La technique « DSAEK » a été difficile à effectuer dans le modèle félin. Une formation rapide de fibrine a été observée dans tous les cas DSAEK (n = 5). 3) Finalement, la fonctionnalité in vivo des endothélia cornéens reconstruits FECD a été évaluée (n = 7). Les évaluations in vivo comprenaient la transparence, la pachymétrie et la tomographie par cohérence optique. Les évaluations post-mortem incluaient la morphométrie des cellules endothéliales, la microscopie électronique à transmission et des immunomarquage de protéines liées à la fonctionnalité. Après la transplantation, la pachymétrie a progressivement diminué et la transparence a progressivement augmenté. Sept jours après la transplantation, 6 des 7 greffes étaient claires. La microscopie électronique à transmission a montré la présence de matériel fibrillaire sous-endothélial dans toutes les greffes d’endothelia reconstruits FECD. Les endothélia reconstruits exprimaient aussi des protéines Na+-K+/ATPase et Na+/HCO3-. En résumé, cette thèse démontre que les cellules endothéliales de la cornée à un stade avancé FECD peuvent être utilisées pour reconstruire un endothélium cornéen par génie tissulaire. La kératoplastie pénétrante a été démontrée comme étant la procédure la plus appropriée pour transplanter ces tissus reconstruits dans l’œil du modèle animal félin. La restauration de l'épaisseur cornéenne et de la transparence démontrent que les greffons reconstruits FECD sont fonctionnels in vivo. Ces nouveaux modèles FECD démontrent une réhabilitation des cellules FECD, permettant d’utiliser le génie tissulaire pour reconstruire des endothelia fonctionnels à partir de cellules dystrophiques. Les applications potentielles sont nombreuses, y compris des études physiopathologiques et pharmacologiques.<br>Fuchs endothelial corneal dystrophy (FECD) is a primary disease of the corneal endothelium. Its pathogenesis is poorly understood. No medical treatment is effective. Surgical treatment (the only available treatment) carries 10% of immunogenic rejection. Experimental models are needed in order to better understand the disease and to investigate potential autologous treatments (to prevent immunogenic rejection). The overall goal of this thesis is to develop an experimental model for FECD using tissue engineering. This was achieved in three steps. 1) An in vitro tissue-engineered FECD model was created and characterized. Briefly, Descemet’s membranes from patients with late-stage FECD undergoing Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK) were used to isolate and culture FECD endothelial cells. Second or third-passaged FECD endothelial cells were seeded on a previously decellularized human cornea. After 2 weeks in culture, TE-FECD corneas (n=6) were assessed using histology, transmission electron microscopy (TEM) and immunofluorescence labeling of various proteins. TE-FECD endothelium yielded a monolayer of polygonal cells well adhered to Descemet’s membrane. The TE-FECD corneal endothelium expressed the function-related proteins Na+-K+/ATPase α1 and Na+/HCO3-. Clusterin expression was faint and uniform. 2) In order to determine the best surgical procedure to transplant the TE-FECD corneas in the feline model, a DSAEK procedure was evaluated and compared to penetrating keratoplasty technique. DSAEK assessments included surgical challenges and clinical outcomes. DSAEK technique was challenging to perform in the feline model. Rapid fibrin formation was observed in all DSAEK cases (n=5). 3) The in vivo functionality of the TE-FECD corneas was assessed. TE-FECD corneas were grafted in the feline model (n=7) using penetrating keratoplasty procedure and observed for seven days. In vivo assessments included transparency, pachymetry, optical coherence tomography, endothelial cell morphometry, TEM and immunostaining of function-related proteins. After transplantation, pachymetry gradually decreased and transparency gradually increased. Seven days after transplantation, 6 out of 7 grafts were clear. Post-mortem TEM showed subendothelial loose fibrillar material deposition in all TE-FECD grafts. The TE grafted endothelium expressed Na+-K+/ATPase and Na+/HCO3-. This thesis demonstrates that endothelial cells from late-stage FECD corneas can be used to engineer a corneal endothelium. Compared to DSEAK, penetrating keratoplasty is a more appropriate procedure for corneal transplantation in the feline model, since the DSAEK procedure in the feline model presently yields inconsistent clinical results. Restoration of corneal thickness and transparency demonstrates that the TE-FECD grafts are functional in vivo. This novel FECD living model suggests a potential role of tissue engineering for FECD cell rehabilitation. Potential applications are numerous, including pathophysiological and pharmacological studies.
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Jui-Yang, Lai. "Functional Biomaterials for Human Corneal Endothelial Cell Sheet Engineering and Regenerative Medicine." 2006. http://www.cetd.com.tw/ec/thesisdetail.aspx?etdun=U0016-1303200709270613.
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Lai, Jui-Yang, and 賴瑞陽. "Functional Biomaterials for Human Corneal Endothelial Cell Sheet Engineering and Regenerative Medicine." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/09808929801336460519.
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博士<br>國立清華大學<br>化學工程學系<br>94<br>Human corneal endothelium in vivo demonstrates an age-related decrease in cell density and cannot be compensated due to its limited regenerative capacity. When the cell density is less than a critical level of 1000 cells/mm2, the endothelial monolayer no longer functions, causing corneal edema and loss of visual acuity. Penetrating keratoplasty (PK) is currently the common way to treat corneas that are opacified due to endothelial dysfunction. However, insufficient supplies of donor corneas and several complications associated with PK remain a worldwide problem. Therefore, transplantation of in vitro cultured human corneal endothelial cells (HCECs) to replace damaged corneal endothelium alone is a promising alternative to PK. In this study, we developed a novel therapy technique to fabricate and transplant cultured HCEC sheets for corneal endothelial reconstruction.
On the basis of plasma chemistry, we have designed a two-step method to prepare a thermo-responsive poly(N-isopropylacrylamide) (PNIPAAm)-grafted culture surface for controlling HCEC adhesion and detachment via a thermal stimulus. The results of surface characterization including energy-dispersive X-ray spectroscopy (EDX), attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), atomic force microscopy (AFM), and static contact angle measurements show that an optimal grafting amount of PNIPAAm is 1.6 μg/cm2. We have also demonstrated that the introduction of AAc segment as short spacers onto the culture support can accelerate the cell detachment, which is beneficial to protect these harvested cells from functional damage.
We further investigated whether the bioengineered HCEC sheets harvested from thermo-responsive culture supports could be used as biological tissue equivalents. Untransformed adult HCECs derived from eye bank corneas were cultivated on PNIPAAm-grafted surfaces for 3 weeks at 37°C. Confluent cell cultures were detached as a laminated sheet by lowering culture temperature to 20°C. In vitro characteristics of HCEC sheets were evaluated by viability, scanning electron microscopy, immunohistochemistry, and histology. Similar to the native corneal endothelium from eye bank donors, the fabricated HCEC monolayers having normal morphology, structure and viability are suitable to be used as tissue replacements for transplantation.
Because of the soft and fragile nature of bioengineered HCEC sheets, we have designed and developed a multi-functional hydrogel carrier system for intraocular delivery of these sheet grafts. The functionality of gamma-sterilized cell carriers made from raw gelatins with a different isoelectric point (IEP = 5.0 and 9.0) and a molecular weight (MW) range from 3 to 100 kDa, was investigated by the determination of mechanical properties, water content, dissolution degree, and cytocompatibility. The results of our study indicate that the gamma-sterilized hydrogel discs consisting of raw gelatins (IEP = 5.0, MW = 100 kDa) are promising candidates as cell sheet carriers for effective corneal endothelial cell transplantation and therapy.
In the in vivo tests, we evaluated the feasibility of HCEC transplantation by harvesting the cell sheets from the thermo-responsive culture supports and delivering with multi-functional gelatin hydrogel discs in a rabbit model. We have shown that the transplanted HCEC sheets could be integrated into rabbit corneas denuded of endothelium. Additionally, when endothelium alone was removed, the rabbit corneas became cloudy and remained opaque throughout the course of the experiment. Once receiving tissue-engineered HCEC sheets, the corneas have returned to a nearly normal thickness. These results imply the biological function of transplanted cell sheets. Our findings indicated that a well-organized and functional HCEC sheet is feasible to be used as tissue equivalents for replacing compromised endothelium.
In the present study, we have demonstrated that the bioengineered human corneal endothelium fabricated from thermo-responsive culture supports and delivered by multi-functional hydrogel carriers can potentially offer a new therapeutic strategy for corneal endothelial cell loss. In addition, functional biomaterials have great potential for development of HCEC sheet engineering and regenerative medicine in ophthalmology. We hope this work will lead to insights into cell sheet-based therapy for corneal endothelial dysfunction and will open an exciting new door to the future.
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Lu, Pei-Lin, and 盧佩琳. "Biopolymer-Based Hydrogel Carriers as Multifunctional Delivery System in Corneal Endothelial Cell Sheet Engineering." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/61945481128087403007.
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博士<br>國立清華大學<br>化學工程學系<br>95<br>Human corneal endothelium in vivo demonstrates an age-related decrease in cell density and cannot be compensated due to its limited regenerative capacity. When the cell density is less than a critical level of 1000 cells/mm2, the endothelial monolayer no longer functions, causing corneal edema and loss of visual acuity. Penetrating keratoplasty (PK) is currently the common way to treat corneas that are opacified due to endothelial dysfunction. However, insufficient supplies of donor corneas and several complications associated with PK remain a worldwide problem. Therefore, transplantation of tissue-engineered human corneal endothelial cell (HCEC) sheets to replace damaged corneal endothelium alone is a promising alternative to PK. This work is devoted to overcome the limitations of cell sheet transplantation.
Because of the soft and fragile nature of bioengineered HCEC sheets, we have designed and developed a multifunctional hydrogel carrier system for intraocular delivery of these sheet grafts. The functionality of gamma-sterilized cell carriers made from raw gelatins with a different isoelectric point (IEP = 5.0 and 9.0) and a molecular weight (MW) range from 3 to 100 kDa, was investigated by the determination of mechanical properties, water content, dissolution degree, and cytocompatibility. The results of our study indicate that the gamma-sterilized hydrogel discs consisting of raw gelatins (IEP = 5.0, MW = 100 kDa) are promising candidates as cell sheet carriers for effective corneal endothelial cell transplantation and therapy.
Development of alternative biomaterials to bovine-based gelatin vehicles can potentially eliminate the risk of bovine spongiform encephalopathy. To investigate whether it was appropriate for use as cell sheet delivery vehicles, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) cross-linked hyaluronic acid (HA) hydrogels were studied by determinations of morphological characteristic, mechanical and thermal property, water content, in vitro degradability, and cytocompatibility. Glutaraldehyde (GTA) cross-linked HA samples were used for comparison. It is concluded that EDC can be successfully applied for HA cross-linking to fabricate structurally stable, mechanically reinforced, readily deformable, transparent, and cytocompatible HA hydrogel discs with the potential to be applied as delivery vehicles for corneal endothelial cell therapy.
On the other hand, intraocular implantation in the anterior chamber has received much attention for the determination of the interactions between the immune privileged tissues and biomaterial implants. A novel methodology based on the anterior chamber of rabbit eyes model was developed to evaluate the in vivo biocompatibility of biomaterials in an immune privileged site. The 7-mm-diameter membrane implants made from either a biological tissue material (amniotic membrane, AM group) or a biomedical polymeric material (gelatin, GM group) were inserted in rabbit anterior chamber for 36 months and characterized by biomicroscopic examinations, intraocular pressure measurements, and corneal thickness measurements. The noninvasive ophthalmic parameters were scored to provide a quantitative grading system. Our data suggest that the anterior chamber of rabbit eyes model is an efficient method for noninvasively determining the immune privileged tissue/biomaterial interactions.
In the present study, we have demonstrated that the multifunctional carrier system is beneficial for transportation and surgical handling of bioengineered human corneal endothelium. Cell sheet transplantation with biopolymer-based hydrogels can potentially offer a new therapeutic strategy for corneal endothelial cell loss. In addition, a novel methodology based on the anterior chamber of rabbit eyes model has great potential for evaluating the ocular biocompatibility and safety of biomaterial carriers. We hope this work will lead to insights into cell sheet-based therapy for ocular regenerative medicine and will open an exciting new door to the future.
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LI, HONG-ZHI, and 李弘志. "The effects ofepidermal growth factor and chondroitin sulfate on the growth of animal corneal endothelial cell culture." Thesis, 1990. http://ndltd.ncl.edu.tw/handle/79084368524196507807.
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"A study of the effects of extracellular matrix on integrin function and human corneal endothelial cell adhesion and proliferation." Tulane University, 2004.
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The human corneal endothelium is a cell layer with a limited regenerative capacity that relies primarily upon the enlargement and migration of existing cells to compensate for any cells lost as the result of injury to the tissue. The limited availability of human corneas and the limited regenerative capacity of the endothelium have led to attempts to induce human corneal endothelial (HCE) cells to proliferate in vivo and restore a functional endothelium Integrins are receptors for extracellular matrix proteins and mediate a variety of biological functions, including differentiation and proliferation. The present study characterized integrin expression and function in cultured HCE cells and a virally-transduced HCE cell strain with a high regenerative capacity. Integrin expression in cultured HCE cells differed somewhat from the reported subunit expression in the intact endothelium. Adhesion studies indicated that E6/E7 cells show a much greater affinity for collagen I than HCE cells Complex matrix and growth factors have been shown to induce proliferation in HCE cells. Tissue culture plastic and several purified matrix proteins were tested, using four different methods, for their ability to promote cell accumulation. A dye conversion assay proved to be unreliable in assessing proliferation. DNA and particle impedance assays indicated that of the purified matrix proteins, laminin and collagen I supported the highest levels of cell accumulation. However, no individual matrix protein was able to come close to the level of cell accumulation induced by matrix and growth factors. Flow cytometry studies indicated that those substrates supporting the highest level of cell accumulation actually induced the fewest number of cells to enter the cell cycle. This finding supports a theory in which only a small population of cells within the endothelium has the ability to divide and replenish lost cells Protein kinase C has been implicated as a role player in a variety of integrin and growth factor signal transduction pathways. HCE cells have the ability to express a variety of PKC isozymes, including PKCalpha, beta I, delta, eta, zeta and iota/lambda. The only commonality among all samples analyzed was the detection of the PKCalpha isozyme, indicating its importance in HCE and E6/E7 signaling pathways<br>acase@tulane.edu
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Shieh, Meng-Chang, and 謝孟璋. "Construction of a mathematical model of electronegative low-density lipoprotein induced vascular endothelial cell dysfunction." Thesis, 2019. http://ndltd.ncl.edu.tw/handle/6kb548.
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碩士<br>國立中山大學<br>醫學科技研究所<br>107<br>The vascular endothelial-cell simulation model had proposed in our previous studies. The study focused on internal voltage and ion clamp experiments in various elements of the model. These experiments confirmed the characteristics of the internal parts of the mathematical model and the calcium ion signal simulation conditions of the intact endothelial cell model. Although, the architecture of the electrophysiological model was established in intracellular clamp experiments, it was not clamped against extracellular ionic conditions to understand the cellular response to external stimuli. In this study, extracellular potassium ion concentration clamp experiments were used to stimulate different Cl-type and K-type endothelial cell models. In this experiment we found that significant depolarization occurs when the endothelial cell model of 100% GVRAC is clamped with approximately 10 mM of external potassium ions. In addition, other low-intensity GVRAC endothelial cell models produce varying degrees of membrane potential stimuli depolarization when clamped to extracellular potassium concentrations below 5 mM. The mathematical model also has a certain degree of ability to simulate the electrophysiological phenomena that occur in actual experiments in the process of simulating actual cases.
In the second phase of the study, we established a superoxide synthesis pathway led by lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) based on this endothelial cell electrophysiological model, and set up a PAF-R pathway that mimics the stimulation of calcium signaling. The mathematical model was able to simulate endothelial nitric oxide and superoxide biosynthesis changes in endothelial dysfunction induced by oxidized low-density lipoprotein (oxLDL) stimulation. The above biosynthesis mechanism is used to simulate the decrease of intracellular nitric oxide utilization rate when nitric oxide and superoxide are combined to synthesize Nitrite, and the independent endothelial cell apoptosis model established in this study can be used for comparison. Combine the results of the above studies to explore the concentration-time relationship between the various elements of these new pathways.
Through this model of endothelial dysfunction, we hope to improve the content that has not yet been added, and to explore the biological pathway in another dimension to gain a deeper understanding of the relevance of each component. We hope that this study will be helpful for pharmacological research.
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Chen, Li Mei, та 陳麗美. "Effects of cell seeding density and transforming growth factor-β challenge on the fabrication of bioengineered corneal keratocyte spheroids". Thesis, 2014. http://ndltd.ncl.edu.tw/handle/vha7vp.
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Chu, Chih-Sheng, and 朱志生. "Electronegative Low Density Lipoprotein Potentiates Vascular Endothelial Cell Toxicity via L5/LOX-1/CRP Cyclic Mechanism on Atherogenesis." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/45921231487731181797.
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博士<br>高雄醫學大學<br>醫學研究所<br>102<br>OBJECTIVES Increased plasma C-reactive protein (CRP) levels are associated with the occurrence and severity of acute coronary syndrome. We investigated whether CRP can be generated in vascular endothelial cells (ECs) after exposure to the most electronegative subfraction of low-density lipoprotein (LDL), L5, which is atherogenic to ECs. Because L5 and CRP are both ligands for the lectin-like oxidized LDL receptor-1 (LOX-1), we also examined the role of LOX-1. The series changes of TNF-a, the upstream mediator of CRP, and of nitrotyrosine were both examined in pre-diabetes patients for the association with coronary atherosclerosis.
METHODS AND RESULTS Plasma LDL samples isolated from asymptomatic hypercholesterolemic (LDL cholesterol [LDL-C] levels, 154.6±20 mg/dL; n = 7) patients and normocholesterolemic (LDL-C levels, 86.1±21 mg/dL; P<0.001; n = 7) control individuals were chromatographically resolved into 5 subfractions, L1-L5. The L5 percentage (L5%) and the plasma L5 concentration ([L5] = L5% × LDL-C) in the patient and control groups were 8.1±2% vs. 2.3±1% (P<0.001) and 12.6±4 mg/dL vs. 1.9±1 mg/dL (P<0.001), respectively. In hypercholesterolemic patients treated with atorvastatin for 6 months (10 mg/day), [L5] decreased from 12.6±4 mg/dL to 4.5±1.1 mg/dL (P = 0.011; n = 5), whereas both [L5] and L5% returned to baseline levels in 2 noncompliant patients 3 months after discontinuation. In cultured human aortic ECs (HAECs), L5 upregulated CRP expression in a dose- and time-dependent manner up to 2.5-fold (P<0.01), whereas the least electronegative subfraction, L1, had no effect. DiI-labeled L1, internalized through the LDL receptor, became visible inside HAECs within 30 seconds. In contrast, DiI-labeled L5, internalized through LOX-1, became apparent after 5 minutes. L5-induced CRP expression manifested at 30 minutes and was attenuated by neutralizing LOX-1. After 30 minutes, L5 but not L1 induced reactive oxygen species (ROS) production. Both L5-induced ROS and CRP production were attenuated by ROS inhibitor N-acetyl cysteine. In 75g OGTT test, post-challenge changes of both TNF-a and nitrotyrosine in pre-diabetes were significantly increased in those with CAD.
CONCLUSIONS Our results suggest that CRP, L5, and LOX-1 form a cyclic mechanism in atherogenesis and that reducing plasma L5 levels with atorvastatin disrupts the vascular toxicity of L5. Post-challenge changes of TNF-a and nitrotyrosine were both shown to be associated with the presence of coronary atherosclerosis in patients with pre-diabetes.
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"Development of a novel bioassay chamber to optimize autologous endothelial cell viability and density on topological and topographical substrates." STATE UNIVERSITY OF NEW YORK AT STONY BROOK, 2009. http://pqdtopen.proquest.com/#viewpdf?dispub=3334946.
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Bostan, Cristina. "Fonctionnalité in vivo d’un endothélium cornéen reconstitué par injection de cellules endothéliales cornéennes dans la chambre antérieure d’un modèle félin." Thèse, 2015. http://hdl.handle.net/1866/16255.
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Introduction : Malgré leur état non-prolifératif in vivo, les cellules endothéliales cornéennes (CEC) peuvent être amplifiées in vitro. Leur transplantation subséquente par injection intracamérale pourrait surmonter la pénurie de tissus associée à l’allo-greffe traditionnelle – l’unique traitement définitif disponible pour les endothéliopathies cornéennes.
Objectif : Évaluer la fonctionnalité d’un endothélium cornéen reconstitué par injection de CEC dans la chambre antérieure du félin. Méthodes : Les yeux droits de 16 animaux ont été opérés. Huit ont été désendothélialisés centralement avec injection de 2x10e5 (n=4) ou 1x10e6 (n=4) CEC félines supplémentées avec Y-27632 et marquées avec SP-DiOC18(3). Deux ont été désendothélialisés complètement et injectés avec 1x10e6 CEC et Y-27632. Six contrôles ont été désendothélialisés centralement (n=3) ou complètement (n=3) et injectés avec Y-27632 sans CEC. La performance clinique, l’intégrité anatomique, le phénotype fonctionnel et l’expression de SP-DiOC18(3) du nouvel endothélium ont été étudiés.
Résultats : Les cornées greffées avec 2x10e5 CEC et les contrôles désendothélialisés centralement ont réussi le mieux cliniquement. Les contrôles désendothélialisés complètement sont restés opaques. L’histopathologie a révélé une monocouche endothéliale fonctionnelle dans les cornées greffées avec 2x10e5 CEC et les contrôles désendothélialisés centralement, une multicouche endothéliale non-fonctionnelle dans les cornées désendothélialisées centralement et greffées avec 1x10e6 CEC, et un endothélium fibrotique non-fonctionnel dans les cornées désendothélialisées complètement. L’expression de SP-DiOC18(3) était rare dans les greffes.
Conclusion : La thérapie par injection cellulaire a reconstitué un endothélium partiellement fonctionnel, auquel les CEC injectées n’ont contribué que peu. L’injection de Y-27632 sans CEC a reconstitué l’endothélium le plus sain. Des études additionnelles investiguant l’effet thérapeutique de Y-27632 seul sont justifiées.<br>Introduction : Despite their growth arrest in vivo, corneal endothelial cells (CEC) can be amplified in vitro. Their subsequent transplantation by cell-injection therapy could overcome the tissue scarcity associated with traditional allo-transplantation, which is the only currently available treatment for irreversible corneal endothelial failure.
Objective : To evaluate the functionality of a corneal endothelium reconstituted by cell- injection therapy in the feline.
Methods: The right eyes of 16 animals underwent surgery. Eight underwent central endothelial scraping and injection with 2x10e5 (n=4) or 1x10e6 (n=4) feline CEC supplemented with Y-27632 and labeled with SP-DiOC18(3). After total scraping, two eyes were injected with 1x10e6 labeled CEC and Y-27632. The central (n=3) or entire (n=3) endothelium was scraped in six controls followed by Y-27632 injection without CEC. Outcomes included clinical performance, anatomical integrity, functional phenotype and SP-DiOC18(3) expression of the new endothelium.
Results: Corneas grafted with 2x10e5 CEC and centrally scraped controls performed the best clinically. Entirely scraped controls remained hazy and thick. Histopathology revealed a confluent, functional endothelial monolayer in corneas grafted with 2x10e5 CEC and centrally scraped controls, a non-uniform, non-functional endothelial multilayer in centrally scraped corneas grafted with 1x10e6 CEC, and a non-functional fibrotic endothelium in entirely scraped grafts and controls. SP-DiOC18(3) was scarce in grafts and absent in controls.
Conclusion : Cell-injection therapy reconstituted an incompletely functional endothelium, to which injected CEC contributed little. Y-27632 injection without CEC reconstituted the healthiest endothelium. Further studies investigating the therapeutic effect of Y-27632 alone are warranted.
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Queirós, Henrique Coimbra de Lemos. "Resultados a longo prazo de lentes intra-oculares fáquicas de fixação à iris: segurança visual e endotelial." Master's thesis, 2020. http://hdl.handle.net/10316/97634.
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Trabalho Final do Mestrado Integrado em Medicina apresentado à Faculdade de Medicina<br>IntroduçãoAs lentes intraoculares fáquicas (pIOL), independentemente da classe, têm excelentes resultados refrativos associados a baixas taxas de complicações intra-operatórias e durante o pós-operatório imediato. As orientações atuais sobre os critérios de implantação e os protocolos de seguimento são baseados em consensos de especialistas. Para além disso, existe uma carência de estudos na literatura com seguimentos prolongados, de forma a avaliar o perfil de segurança endotelial. Assim, este trabalho propõe caracterizar uma coorte retrospetiva, com um período de seguimento mínimo de 5 anos, monitorizando a evolução da contagem de células endoteliais (ECD) após implantação de lentes fáquicas rígidas ou flexíveis.MétodosEstudo retrospetivo que inclui 124 olhos de 73 doentes submetidos a cirurgia refrativa com lentes fáquicas rígidas (Artisan Myopia and Hyperopia, Ophtec BV; Verisyse, Abbott Medical Optics) ou flexíveis (Artiflex Myopia, Ophtec BV; Veriflex (Abbott Medical Optics)), com um follow-up mínimo de 5 anos e profundidade da câmara anterior (endoACD) mínima de 2.8 mm. Os preditores da perda de células endoteliais (EC) foram avaliados com recurso a regressões lineares. Para calcular o índice de segurança utilizou-se o rácio entre a média da acuidade visual corrigida (DCVA) pós-operatória (na visita dos 5 anos) e a média da DCVA pré-operatória.ResultadosForam incluídos 51 olhos no grupo das lentes rígidas (tempo de seguimento médio de 7.3 anos) e 73 olhos no grupo das lentes flexíveis (tempo de seguimento médio de 6.8 anos). A perda anual de células endoteliais foi de 53.47 células/mm2 (-1.84%/ano) no grupo das lentes rígidas e de 35.05 células/mm2 (-1.28%/ano) no grupo das lentes flexíveis, o que demonstra uma diferença estatisticamente significativa entre os dois grupos (p= 0.050). No grupo das lentes rígidas, uma menor profundidade de câmara anterior bem como um aumento absoluto nas contagens pré-operatórias das células endoteliais foram preditores significativos e independentes de uma maior perda de EC de acordo com um modelo de regressão linear bivariada. (p= 0.002 e p= 0.001, respetivamente). Contudo, dentro do grupo das lentes flexíveis, essa associação apenas foi encontrada para as contagens pré-operatórias (p= 0.009), e não para a profundidade de câmara anterior (p= 0.242). O índice de segurança foi de 1.32±0.32 e 1.16±0.28 para as lentes rígidas e flexíveis, respetivamente.DiscussãoNa coorte retrospetiva estudada, foram encontradas perdas de células endoteliais similares ou superiores à perda fisiológica esperada, em ambos os grupos analisados (lentes fáquicas flexíveis e rígidas). Enquanto uma contagem superior de células endoteliais pré-operatória está associada a uma maior perda de EC em ambos os grupos, uma menor profundidade da câmara anterior foi apenas significativa no grupo das lentes rígidas. Isto sugere que variações pré-operatórias da profundidade da câmara anterior (sempre acima dos 2.8 mm necessários para a implantação) podem não influenciar significativamente a perda de EC em lentes flexíveis, ao contrário do que se verifica nas lentes rígidas. Ambos os grupos apresentam índices de segurança superiores à unidade ao fim de 5 anos, para todos os olhos.<br>Introduction Phakic intraocular lenses (pIOLs), regardless of type, have excellent refractive results with a low rate of intra and early post-operative complications. Current guidelines for implantation criteria and post-operative management were agreed upon specialist consensus. However long-term follow-up studies are still lacking to better evaluate endothelial safety profile. We purpose to investigate long-term (5-year) endothelial cell density (ECD) changes following implantation of iris-fixated phakic intraocular lenses, rigid and flexible.Methods Retrospective study including 124 eyes of 73 patients receiving pIOLs, either rigid (Artisan Myopia and Hyperopia, Ophtec BV; Verisyse, Abbott Medical Optics) or flexible (Artiflex Myopia, Ophtec BV; Veriflex (Abbott Medical Optics)), with a minimum follow-up of 5 years, all implanted with a minimum endothelial anterior chamber depth (endoACD) of 2.8 mm. Predictors of endothelial cell (EC) loss were evaluated using linear regression analysis. The best corrected visual acuity (DCVA) at the 5-year visit was used to calculate the safety index (post-operative DCVA / pre-operative DCVA).ResultsWe included 51 eyes in the rigid pIOL group (mean follow-up of 7.3 years) and 73 eyes in the flexible pIOL group (mean follow-up 6.7 years). The annualized EC loss was 53.47 cells/mm2 (-1.8%/year) in the rigid pIOL group and 35.05 cells/mm2 (-1.3%/year) in the flexible pIOL group, showing statistically difference between groups (p= 0.050). In the rigid pIOL group, increased absolute preoperative ECD and decreased endoACD were both significant and independent predictors of a higher EC loss rate in a bivariate linear regression model (p= 0.001 and p= 0.002, respectively). Interestingly, in the flexible pIOL group, increased absolute preoperative ECD, but not deceased endoACD, predicted increased EC loss rate in univariate regression analysis (p= 0.009 and p= 0.242, respectively). The safety index was 1.32±0.32 (range 1.00-2.00) and 1.16±0.28 (range 0.80-2.00) in the rigid and flexible group, respectively. DiscussionSimilar or higher than physiologic annualized EC loss rates were found in both groups in a long-term follow-up cohort. While increased preoperative ECD predicted a higher EC loss rate in both groups, a smaller endoACD was only a significant predictor in the rigid pIOL group. This suggests that variations in preoperative endoACD (always above the 2.8 mm required for implantation) may not significantly influence future EC loss rate in flexible pIOLs to the extent that it appears to do in rigid pIOLs. Both pIOLs types have a high safety index with every eye attaining a safety index larger than 1.00 at 5 years of follow-up.