Proliferative capacity of corneal endothelial cells

The corneal endothelial monolayer helps maintain corneal transparency through its barrier and ionic "pump" functions. This transparency function can become compromised, resulting in a critical loss in endothelial cell density (ECD), corneal edema, bullous keratopathy, and loss of visual acuity. Although penetrating keratoplasty and various forms of endothelial keratoplasty are capable of restoring corneal clarity, they can also have complications requiring re-grafting or other treatments. With the increasing worldwide shortage of donor corneas to be used for keratoplasty, there is a greater need to find new therapies to restore corneal clarity that is lost due to endothelial dysfunction. As a result, researchers have been exploring alternative approaches that could result in the in?vivo induction of transient corneal endothelial cell division or the in?vitro expansion of healthy endothelial cells for corneal bioengineering as treatments to increase ECD and restore visual acuity. This review presents current information regarding the ability of human corneal endothelial cells (HCEC) to divide as a basis for the development of new therapies. Information will be presented on the positive and negative regulation of the cell cycle as background for the studies to be discussed. Results of studies exploring the proliferative capacity of HCEC will be presented and specific conditions that affect the ability of HCEC to divide will be discussed. Methods that have been tested to induce transient proliferation of HCEC will also be presented. This review will discuss the effect of donor age and endothelial topography on relative proliferative capacity of HCEC, as well as explore the role of nuclear oxidative DNA damage in decreasing the relative proliferative capacity of HCEC. Finally, potential new research directions will be discussed that could take advantage of and/or improve the proliferative capacity of these physiologically important cells in order to develop new treatments to restore corneal clarity.     

人角膜内皮细胞增殖潜能的研究进展

体外培养人角膜内皮细胞的成功和人角膜内皮细胞永生系的建立,充分肯定了角膜内皮细胞的增殖能力.细胞周期蛋白的研究证明角膜内皮细胞只是停留在细胞周期的G<,1>期,而不是退出了细胞周期;年轻人角膜内皮细胞增殖性强于老年人,角膜周边部内皮细胞的增殖性强于角膜中央;众多因素参与调节角膜内皮细胞的增殖,抑制因素主要为接触抑制及转化生长因子-β,促进因素主要为细胞因子(EGF、FGF、NGF)及细胞外基质.人角膜内皮前体细胞的成功分离、鉴定及培养为治疗角膜内皮失代偿提供了美好前景.     

Injuries of the corneal endothelium

Examples of "endogenous" or direct and "endogenous" or indirect endothelial trauma are presented. Corneal contusion, McCannel suture, YAG laser iridectomy, congenital luxation of the lens, and chronic over wearing of contact lenses all cause traumatic changes. The etiology and effects of these changes are discussed. Specular microscopy of the corneal endothelium permits good clinical control of both the trauma and wound healing.     

Snailtracks of the corneal endothelium

An unusual pattern of corneal endothelial cell alterations has been observed at the slitlamp in eyes following intraocular surgery. Because of their appearance, we have called these changes "snailtracks". We have commonly seen these snailtracks following extracapsular cataract extraction. Similar changes have been noted in cadaver corneas stored in McCarey-Kaufman (M-K) medium. This study presents information which suggests that these changes represent corneal endothelial cell damage.     

Human corneal endothelium

Summary The infant cornea contains many densely packed cells with large nuclei and little cytoplasm. As the cornea grows during childhood, cell density decreases by almost 50% and the cells become correspondingly larger. The adult cornea maintains a relatively uniform density of hexagonal cells which retain a generally uniform size and appearance. In the aged cornea, however, a pleomorphism develops. The aged endothelial surface is covered by a mixture of enormous as well as very small cells.

---

Zusammenfassung In dem Hornhautendothel des Kindes sind die Zellen dicht gedrängt; ihre Kerne sind groß und das Protoplasma ist gering. Während des Wachstums der kindlichen Hornhaut nimmt die Zelldichte um etwa 50% ab, während die Zellgröße entsprechend zunimmt. Das Hornhautendothel des Erwachsenen zeigt eine relativ einheitliche Zelldichte. Auch die Größe und Morphologie der Zellen ist in diesem Alter im allgemeinen überall gleich. Jedoch ist die senile Hornhaut durch einen Pleomorphismus charakterisiert und an der Oberfläche des Endothels findet sich ein Gemisch von sehr großen und sehr kleinen Zellen.

---

Résumé L'endothélium cornéen infantile contient beaucoup de cellules en groupes compacts avec de larges noyaux et peu de cytoplasme. Pendant la croissance de l'endothélium la densité cellulaire diminue d'environ 50% et les cellules s'élargissent d'une façon correspondante. La cornée adulte conserve une densité relativement uniforme de cellules hexagonales. Il en est de même de leur dimension et de leur apparence générale. Cependant on constate un pléomorphisme dans la cornée âgée. La surface de l'endothélium est constituée chez des personnes de 65 ans et plus par un mélange de cellules énormes et très petites.

---

---

This work was supported by U.S. Public Health Service Grants NB 05037 and NB 08210 from the National Institute of Neurological Diseases and Stroke, National Institutes of Health.     

自体血管内皮细胞移植替代角膜内皮层的实验研究

目的 探讨自体血管内皮细胞(VECs)移植替代角膜内皮层的可行性.方法 40只家兔随机分为4组.A组,家兔VECs培养移植于去后弹力层的白体角膜植片内表面;B组,角膜植片去后弹力层后移植回原植床;C组,角膜植片取下后移植回原植床;D组,家兔不做处理.术后观察VECs生长状况及角膜植片透明度,A型超声测其厚度.结果 A组角膜植片混浊,1周内最明显,后混浊减轻,2周可见前房.细胞生长良好,部分细胞形状不规则.B组角膜植片混浊且不改善.C、D两组角膜无混浊.结论 VECs可替代自体角膜内皮层的屏障功能,维持角膜的透明状态.     

Antimicrobials and the corneal endothelium

The effects of various intraocular antibiotics and preservatives on the rabbit corneal endothelium were evaluated using vital staining, light, scanning, and transmission electron microscopy after topical application of the drugs and up to 2 days following anterior chamber injection. Benzalkonium chloride consistently produced the most severe and probably irreversible damage. Some changes which persisted for 2 days were found with methicillin. Gentamicin produced a moderate amount of cellular edema which disappeared after 2 days. Minimal changes were produced by dexamethasone and amphotericin B. Although these results should not be directly interpolated to the human situation, the face that some of these agents did produce changes in the rabbit corneal endothelium, which is well known to be heartier than human endothelium, indicates that caution should be taken when one considers injecting antibiotics intraocularly.     

Clinical responses of the corneal endothelium

The corneal endothelium maintains corneal deturgescence and clarity by a pump-leak mechanism first described by David Maurice. This cell layer can be investigated clinically with specular microscopy, fluorophotometry, and pachymetry. We describe the clinical responses of the corneal endothelium to aging, drugs, glaucoma, contact lens wear, trauma, disease, and surgery.     

Regeneration of the human corneal endothelium

The ability of the human corneal endothelium to regenerate is studied with the scanning electron microscope through examples of corneal diseases and penetrating keratoplasties. This study does not lead to final conclusions on the possibilities of regeneration of the human corneal endothelium but allows us to say that:

regeneration occurs through size increasing and deformation of the remaining cells.

the increase in number and size of surface microvilli may simply indicate a state of cell activation.

the présence of two nuclei in one cell is probably obtained by amitotic division but no complete mitosis has been seen.

Displacement of endothelial cells is a real progression and the cell is able to overcome obstacles.

the fibroblastic transformation of the endothelial cells is present in man but this may simply represent the migrating form of the cells.

     

Ultraviolet-B damages corneal endothelium

Direct in vivo observation of acute ultraviolet (UV)-induced corneal endothelial damage is not possible due to the more severe damage produced in the epithelium. In order to quantify damage and evaluate endothelial recovery an indirect method was used. Eyes of pigmented rabbits were irradiated with UV-B (290 to 320 nm) isolated from the output of a high-pressure 1000 W reflectorized Xenon arc lamp by a grating monochromator and appropriate filters. The peak wavelength of the radiation used was 305 nm, with a 18 nm bandwidth at half-maximum. Corneal thickness variations measured with a modified Zeiss (Oberkochen) pachometer were used to follow alterations in epithelial and endothelial function. Epithelial damage alone resulted in a maximum thickness increase of 13.5% within 24 hr with recovery within a further 24 hr. Greater increases in corneal thickness, in the absence of anterior uveal involvement, were taken to indicate endothelial damage, and reached maximum at 2 days, with recovery occurring in 7 days. The threshold for endothelial damage sufficient to disturb corneal deturgescence was 0.12 J X cm-2.     

角膜内皮基因转移

基因治疗的关键技术是基因转移。角膜内皮基因转移是角膜内皮基凶治疗的基础,其主要方法有病毒载体和非病毒载体介导法,他们各有其优缺点。对角膜内皮基因转移技术和载体的研究现状,及未来载体的研究方向进行综述。     

Intracameral thrombin and the corneal endothelium

We perfused the endothelia of isolated human corneas mounted in the specular microscope with BSS Plus containing 1,000-U/ml or 100-U/ml dilutions of two commercially available topical thrombin preparations. Corneas perfused with thrombin at 1,000 U/ml showed intracellular and intercellular vacuole formation and altered junctional complexes. As listed on the package inserts, the thrombin preparations contained preservatives and other additives that present a significant osmotic load in 1,000-U/ml preparations. Corneas perfused with 100-U/ml thrombin solutions showed a significant attenuation in their deswelling rate but no ultrastructural alterations. One available thrombin preparation when diluted to 100 U/ml had a glycine concentration associated with previous retinal electroretinography changes. Polyacrylamide gel electrophoresis of one manufacturer's thrombin solution showed multiple high and low molecular weight constituents. Analysis of particulate contamination showed one 100-U/ml thrombin preparation to have a large quantity of particulates. Although thrombin may be useful when applied topically as an aid in surgical hemostasis, its use intraocularly presents substantial concern regarding the preparation's purity, additives, contaminants, and adverse effects on ocular tissues.