Joseph A. Bonanno

A link between tear instability and hyperosmolarity in dry eye.

PURPOSE Tear film instability and tear hyperosmolarity are considered core mechanisms in the development of dry eye. The authors hypothesize that evaporation and instability produce transient shifts in tear hyperosmolarity that lead to chronic epithelial stress, inflammation, and symptoms of ocular irritation. The purpose of this study was to provide indirect evidence of short-term hyperosmolar conditions during tear instability and to test whether the corneal epithelium responds to transient hyperosmolar stress. METHODS Five subjects kept one eye open as long as possible, and overall discomfort and sensations associated with tear break-up were scaled. Later, the same subjects used the same scales to report discomfort sensations after instillation of NaCl and sucrose hyperosmolar drops (300-1000 mOsM/kg). A two-alternative, forced-choice experiment was used to obtain osmolarity thresholds. In the second experiment, primary cultured bovine corneal epithelial cells were transiently stressed with the same range of hyperosmolar culture medium, and proinflammatory mitogen-activated protein kinase (MAPKs) were measured by Western blot analysis. RESULTS Tear instability led to an average discomfort rating of 6.13 and sensations of burning and stinging. These sensations also occurred with hyperosmolar solutions (thresholds, 450-460 mOsM/kg) that required 800 to 900 mOsM/kg to generate the same discomfort levels reported during tear break-up. MAPK was activated at 600 mOsM/kg of transient hyperosmolar stress. CONCLUSIONS These experiments provide a link between hyperosmolarity and tear instability, suggesting that hyperosmolar levels in the tear film may transiently spike during tear instability, resulting in corneal inflammation and triggering sensory neurons.

Re-evaluation of the oxygen diffusion model for predicting minimum contact lens Dk/t values needed to avoid corneal anoxia.

PURPOSE (1) To update Fatts mathematical model of the distribution of oxygen tension (pO2) across the cornea and contact lens (CL) to include the recent finding that corneal oxygen consumption increases with the acidification that occurs with CL wear. (2) To estimate the minimum transmissibility (CL Dk/t) to avoid epithelial anoxia or to avoid stromal anoxia. METHODS A five-layer static and one-dimensional mathematical model of oxygen diffusion through the cornea based on Fatts models was used. The relationships between acidosis and increased QO2, and acidosis and CL Dk/t were used to estimate corneal QO2 for a given CL Dk/t. RESULTS (1) Revised model predictions are in agreement with direct tear pO2 measurements beneath CLs in the rabbit. (2) For the human eye, the minimum CL Dk/t for oxygen delivery to the basal epithelial cells was determined to be 23 for the open eye and 89 for the closed eye. To prevent anoxia throughout the entire corneal thickness the Dk/t requirements are 35 for the open eye and 125 for the closed eye. CONCLUSIONS (1) Model predictions of the oxygen distribution beneath contact lenses are significantly lower than previous models that did not include the effect of acidosis on corneal QO2. (2) Minimum Dk/t values that allow oxygen delivery to the basal epithelium are in agreement with the Dk/t needed to avoid corneal edema.

Identity and regulation of ion transport mechanisms in the corneal endothelium

Corneal transparency is dependent on regulation of the hydration of the corneal stroma. Water is driven into the cornea across the epithelial and endothelial cell layers by the stromal swelling pressure. This fluid leak into the cornea is counterbalanced by the corneal fluid pump, which is predominantly attributed to the ion and fluid transport capacity of the endothelial cell layer. Primary and secondary active transport mechanisms are responsible for generating a net ion flux from the stromal to anterior chamber side of the endothelium; however, the identity and location of all the components of this transport system are not known. The endothelial fluid pump is dependent on the presence of Cl(-) and HCO(3)(-), and can be slowed by carbonic anhydrase inhibitors. A number of anion transport mechanisms have been identified and characterized in the endothelium, including basolateral Na(+)/2HCO(3)(-) cotransport, Na(+)/K(+)/2Cl(-) cotransport, Cl(-)/HCO(3)(-) exchange, and apical anion channels permeable to both Cl(-) and HCO(3)(-). Furthermore, there is evidence for a carbonic anhydrase mediated CO(2)-diffusive mode of apical HCO(3)(-) flux. These findings are incorporated into a new model of transendothelial anion transport, which suggests that there are a number of alternate pathways for anion transport. There have been few studies on activation of signal transduction pathways that could stimulate endothelial fluid transport. Interestingly, recent studies show that multiple autocrine signaling pathways are in place that could be upregulated during physical stimulation and may be responsible for maintaining basal levels of fluid secretion.

Overnight orthokeratology: visual and corneal changes.

Purpose. To achieve an optimal fit with reverse geometry Contex OK lenses and to determine a time course for and the stability of visual and corneal changes in achieving maximal refractive, corneal curvature, and corneal thickness changes after overnight wear of OK B and D series lenses. Methods. This investigation was conducted under an Food and Drug Administration IDE G000059. Both eyes of 10 subjects were fitted with the lenses, and uncorrected visual acuity, refractive correction, contrast sensitivity, corneal curvature, and corneal thickness were measured at baseline and at 1 day, 1week, 1 month, and 3 months after lenses were worn. Except for baseline, data were collected at four different times during the day, immediately following lens removal and 4, 8, and 12 hours after lens removal. Results. The results from eight subjects showed that uncorrected visual acuity, refractive correction, contrast sensitivity, and corneal curvature all changed significantly (P =0.01) overnight. By the end of 1 week, all corneal and visual changes had reached a maximal level and remained fairly stable during the day. These changes were sustained at 3 months. The epithelial thickness data from four subjects showed that the corneal epithelial thickness was reduced by approximately 19 &mgr;m after 3 months of lens wear. Conclusions. Successful fitting of OK B and D series lenses requires a thorough understanding of the lens–cornea relationship. Full effect of overnight orthokeratology is achieved by the end of 1 week. The visual and corneal changes remain stable for all waking hours of the day and allow patients to enjoy excellent device-free vision (20/20).

Calcium influx factor from cytochrome P-450 metabolism and secretion-like coupling mechanisms for capacitative calcium entry in corneal endothelial cells.

Notwithstanding extensive efforts, the mechanism of capacitative calcium entry (CCE) remains unclear. Two seemingly opposed theories have been proposed: secretion-like coupling (Patterson, R. L., van Rossum, D. B., and Gill, D. L. (1999) Cell 98, 487–499) and the calcium influx factor (CIF) (Randriamampita, C., and Tsien, R. Y. (1993)Nature 364, 809–814). In the current study, a combinatorial approach was taken to investigate the mechanism of CCE in corneal endothelial cells. Induction of cytochrome P-450s by β-naphthoflavone (BN) enhanced CCE measured by Sr2+ entry after store depletion. 5,6-Epoxyeicosatrienoic acid (5,6-EET), a proposed CIF generated by cytochrome P-450s (Rzigalinski, B. A., Willoughby, K. A., Hoffman, S. W., Falck, J. R., and Ellis, E. F. (1999) J. Biol. Chem. 274, 175–182), induced Ca2+ entry. Both BN-enhanced CCE and the 5,6-EET-induced Ca2+ entry were inhibited by the CCE blocker 2-aminoethoxydiphenyl borate, indicating a role for cytochrome P-450s in CCE. Treatment with calyculin A (CalyA), which causes condensation of cortical cytoskeleton, inhibited CCE. The actin polymerization inhibitor cytochalasin D partially reversed the inhibition of CCE by CalyA, suggesting a secretion-like coupling mechanism for CCE. However, CalyA could not inhibit CCE in BN-treated cells, and 5,6-EET caused a partial activation of CCE in CalyA-treated cells. These results further support the notion that cytochrome P-450 metabolites may be CIFs. The vesicular transport inhibitor brefeldin A inhibited CCE in both vehicle- and BN-treated cells. Surprisingly, Sr2+ entry in the absence of store depletion was enhanced in BN-treated cells, which was also inhibited by 2-aminoethoxydiphenyl borate. An integrative model suggests that both CIF from cytochrome P-450 metabolism and secretion-like coupling mechanisms play roles in CCE in corneal endothelial cells.

Ion Transport Function of SLC4A11 in Corneal Endothelium

PURPOSE Mutations in SLC4A11, a member of the SLC4 superfamily of bicarbonate transporters, give rise to corneal endothelial cell dystrophies. SLC4A11 is a putative Na⁺ borate and Na⁺:OH⁻ transporter. Therefore we ask whether SLC4A11 in corneal endothelium transports borate (B[OH]₄⁻), bicarbonate (HCO3⁻), or hydroxyl (OH⁻) anions coupled to Na⁺. METHODS SLC4A11 expression in cultured primary bovine corneal endothelial cells (BCECs) was determined by semiquantitative PCR, SDS-PAGE/Western blotting, and immunofluorescence staining. Ion transport function was examined by measuring intracellular pH (pHi) or Na⁺ ([Na⁺](i)) in response to Ringer solutions with/without B(OH)₄⁻ or HCO₃⁻ after overexpressing or small interfering RNA (siRNA) silencing of SLC4A11. RESULTS SLC4A11 is localized to the basolateral membrane in BCEC. B(OH)₄⁻ (2.5-10 mM) in bicarbonate-free Ringer induced a rapid small acidification (0.01 pH unit) followed by alkalinization (0.05-0.1 pH unit), consistent with diffusion of boric acid into the cell followed by B(OH)₄⁻. However, the rate of B(OH)₄⁻-induced pHi change was unaffected by overexpression of SLC4A11. B(OH)₄⁻ did not induce significant changes in resting [Na⁺(i)] or the amplitude and rate of acidification caused by Na⁺ removal. siRNA-mediated knockdown of SLC4A11 (∼70%) did not alter pHi responses to CO₂/HCO₃⁻-rich Ringer, Na⁺-free induced acidification, or the rate of Na⁺ influx in the presence of bicarbonate. However, in the absence of bicarbonate, siSLC4A11 knockdown significantly decreased the rate (43%) and amplitude (48%) of acidification due to Na⁺ removal and recovery (53%) upon add-back. Additionally, the rate of acid recovery following NH₄⁺ prepulse was decreased significantly (27%) by SLC4A11 silencing. CONCLUSIONS In corneal endothelium, SLC4A11 displays robust Na⁺-coupled OH⁻ transport, but does not transport B(OH)₄⁻ or HCO₃⁻.

Oxygen-deficient metabolism and corneal edema

Wear of low-oxygen-transmissible soft contact lenses swells the cornea significantly, even during open eye. Although oxygen-deficient corneal edema is well-documented, a self-consistent quantitative prediction based on the underlying metabolic reactions is not available. We present a biochemical description of the human cornea that quantifies hypoxic swelling through the coupled transport of water, salt, and respiratory metabolites. Aerobic and anaerobic consumption of glucose, as well as acidosis and pH buffering, are incorporated in a seven-layer corneal model (anterior chamber, endothelium, stroma, epithelium, postlens tear film, contact lens, and prelens tear film). Corneal swelling is predicted from coupled transport of water, dissolved salts, and especially metabolites, along with membrane-transport resistances at the endothelium and epithelium. At the endothelium, the Na+/K+ - ATPase electrogenic channel actively transports bicarbonate ion from the stroma into the anterior chamber. As captured by the Kedem-Katchalsky membrane-transport formalism, the active bicarbonate-ion flux provides the driving force for corneal fluid pump-out needed to match the leak-in tendency of the stroma. Increased lactate-ion production during hypoxia osmotically lowers the pump-out rate requiring the stroma to swell to higher water content. Concentration profiles are predicted for glucose, water, oxygen, carbon dioxide, and hydronium, lactate, bicarbonate, sodium, and chloride ions, along with electrostatic potential and pressure profiles. Although the active bicarbonate-ion pump at the endothelium drives bicarbonate into the aqueous humor, we find a net flux of bicarbonate ion into the cornea that safeguards against acidosis. For the first time, we predict corneal swelling upon soft-contact-lens wear from fundamental biophysico-chemical principles. We also successfully predict that hypertonic tear alleviates contact-lens-induced edema.

Central and peripheral corneal swelling accompanying soft lens extended wear.

ABSTRACT Central and peripheral corneal thickness changes were measured after 3 hr of eye closure with and without contact lens wear. Spun cast and lathe cut hydrogel extended wear lenses of −1.25, −6.00, and −9.00 D were used. Each of these conditions produced 25 to 50% less swelling in the peripheral than in the central cornea. Central swelling was greater with higher minus spun cast lenses, but not related to the power of the lathe cut lenses. Higher minus lens powers of both lens types produced greater peripheral swelling. Differences between central and peripheral swelling were smaller with the higher minus lenses, indicating little, if any, averaging of tear oxygen tension under the lens when the eyes are closed. The mechanism resulting in less peripheral corneal swelling is likely to be caused by an anatomical “clamping” at the corneal limbus.

Studies on the expression of mRNA for anion transport related proteins in corneal endothelial cells

Purpose. Chloride and bicarbonate are necessary for maintenance of fluid transport by the corneal endothelium, however there is little information on the identity of anion transport proteins that could serve as anion efflux mechanisms in endothelial cells. Therefore, we ask whether mRNA for the anion transport related proteins, CFTR, CLC-2, ClC-3, ClC-5 and AE2, are expressed in human, bovine or rabbit corneal endothelium. Methods. RT-PCR was performed for CFTR, CLC-2, ClC-3, ClC-5 and AE2 using total RNA from fresh human, bovine and rabbit corneal endothelium as well as cultured bovine corneal endothelial cells (CBCEC). Specificity of PCR products was confirmed by sequencing. Results. RT-PCR analysis gave positive bands at the predicted size for CLC-3 and CLC-5 from fresh human, rabbit and bovine as well as CBCEC. However, for CLC-2, no band was apparent around the predicted size from fresh and cultured corneal endothelium. A band at the predicted size was obtained for CFTR from fresh human, rabbit and bovine endothelium, as well as from CBCEC. RT-PCR analysis for AE2 produced specific bands from fresh human, rabbit and bovine corneal endothelium, but no positive band was obtained from CBCEC. Sequencing analysis further confirmed the identities of CLC-3, CLC-5, CFTR and AE2 in corneal endothelium. Conclusions. CFTR, CLC-3 and ClC-5 are expressed in fresh and cultured corneal endothelial cells. However, consistent with previous immunoblots studies, AE2 is only expressed in fresh corneal endothelium. These results have implications for modeling possible apical anion efflux mechanisms in corneal endothelium.

Telomerase Immortalization of Human Corneal Endothelial Cells Yields Functional Hexagonal Monolayers

Human corneal endothelial cells (HCEnCs) form a monolayer of hexagonal cells whose main function is to maintain corneal clarity by regulating corneal hydration. HCEnCs are derived from neural crest and are arrested in the post-mitotic state. Thus cell loss due to aging or corneal endothelial disorders leads to corneal edema and blindness–the leading indication for corneal transplantation. Here we show the existence of morphologically distinct subpopulations of HCEnCs that are interspersed among primary cells and exhibit enhanced self-renewal competence and lack of phenotypic signs of cellular senescence. Colonies of these uniform and hexagonal HCEnCs (HCEnC-21) were selectively isolated and demonstrated high proliferative potential that was dependent on endogenous upregulation of telomerase and cyclin D/CDK4. Further transduction of HCEnC-21 with telomerase yielded a highly proliferative corneal endothelial cell line (HCEnT-21T) that was devoid of oncogenic transformation and retained critical corneal endothelial cell characteristics and functionality. This study will significantly impact the fields of corneal cell biology and regenerative medicine.