Academic literature on the topic 'Corneal endothelial cell density'

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.

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.

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.

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.

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

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.

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

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.