Corneal Edema in Inducible Knockout Is Initiated by Mitochondrial Superoxide Induced Src Kinase Activation.

Purpose: Inducible KO leads to corneal edema by disruption of the pump and barrier functions of the corneal endothelium (CE). The loss of Slc4a11 NH-activated mitochondrial uncoupling leads to mitochondrial membrane potential hyperpolarization-induced oxidative stress. The goal of this study was to investigate the link between oxidative stress and the failure of pump and barrier functions and to test different approaches to revert the process.

Nanoliposomes for Sensing Local Osmolarity of the Tear Film on the Corneal Surface.

Local hotspots of elevated tear hyperosmolarity (exceeding 900 mOsM) are predicted in dry eye disease (DED) but have not been measured. This study aims to develop, characterize, and evaluate the suitability of fluorescent nanoliposomes for noninvasive sensing of the local osmolarity of the tear film. Fluorescent nanoliposomes, loaded with calcein (susceptible to self-quenching; sensor dye) and sulforhodamine 101 (SR101; reference dye), were produced by the thin-film hydration method.

Rescue of the Congenital Hereditary Endothelial Dystrophy Mouse Model by Adeno-Associated Viruse-Mediated Replacement.

Purpose: Congenital hereditary endothelial dystrophy (CHED) is a rare condition that manifests at an early age showing corneal edema, increased oxidative stress, mitochondrial dysfunction, and eventually apoptosis of the endothelium due to loss of function of the membrane transport protein SLC4A11. This project tested whether replacing into the CHED mouse model can reverse the disease-associated phenotypes.

Design: Experimental study.

Mitochondrial ROS in KO Corneal Endothelial Cells Lead to ER Stress.

Recent studies from mice have identified glutamine-induced mitochondrial dysfunction as a significant contributor toward oxidative stress, impaired lysosomal function, aberrant autophagy, and cell death in this Congenital Hereditary Endothelial Dystrophy (CHED) model. Because lysosomes are derived from endoplasmic reticulum (ER)-Golgi, we asked whether ER function is affected by mitochondrial ROS in KO corneal endothelial cells. In mouse corneal endothelial tissue, we observed the presence of dilated ER and elevated expression of ER stress markers BIP and CHOP.

The H Transporter SLC4A11: Roles in Metabolism, Oxidative Stress and Mitochondrial Uncoupling.

Solute-linked cotransporter, SLC4A11, a member of the bicarbonate transporter family, is an electrogenic H transporter activated by NH and alkaline pH. Although SLC4A11 does not transport bicarbonate, it shares many properties with other members of the SLC4 family. SLC4A11 mutations can lead to corneal endothelial dystrophy and hearing deficits that are recapitulated in SLC4A11 knock-out mice.

RNA sequencing uncovers alterations in corneal endothelial metabolism, pump and barrier functions of Slc4a11 KO mice.

Slc4a11 KO mice show significant corneal edema, altered endothelial morphology, and mitochondrial ROS at an early age without a decrease in endothelial cell density. We examined the differential gene expression profile between wild type (WT) and KO with the goal of finding pathways related to corneal endothelial metabolic, pump and barrier function that can explain the corneal edema. Freshly dissected Corneal Endothelium-Descemet's Membrane (CEDM) and cultured Mouse Corneal Endothelial Cells (MCEC) were obtained from WT and Slc4a11 KO mice.

Mitochondrial ROS Induced Lysosomal Dysfunction and Autophagy Impairment in an Animal Model of Congenital Hereditary Endothelial Dystrophy.

Purpose: The Slc4a11 knock out (KO) mouse model recapitulates the human disease phenotype associated with congenital hereditary endothelial dystrophy (CHED). Increased mitochondrial reactive oxygen species (ROS) in the Slc4a11 KO mouse model is a major cause of edema and endothelial cell loss. Here, we asked if autophagy was activated by ROS in the KO mice.

Mitochondrial Targeting of the Ammonia-Sensitive Uncoupler SLC4A11 by the Chaperone-Mediated Carrier Pathway in Corneal Endothelium.

Purpose: SLC4A11, an electrogenic H+ transporter, is found in the plasma membrane and mitochondria of corneal endothelium. However, the underlying mechanism of SLC4A11 targeting to mitochondria is unknown.

Methods: The presence of mitochondrial targeting sequences was examined using in silico mitochondrial proteomic analyses.

Inducible Slc4a11 Knockout Triggers Corneal Edema Through Perturbation of Corneal Endothelial Pump.

Purpose: The conventional Slc4a11 knockout (KO) shows significant corneal edema at eye opening, a fact that complicates the study of the initial events leading to edema. An inducible KO would provide opportunities to examine early events following loss of Slc4a11 activity.

Methods: Slc4a11 Flox (SF) mice were crossed with mice expressing the estrogen receptor Cre Recombinase fusion protein and fed tamoxifen (Tm) for two weeks.

Bicarbonate activates glycolysis and lactate production in corneal endothelial cells by increased pH.

Recent studies have shown that lactate coupled water flux is the underlying mechanism of the corneal endothelial pump, which is highly dependent on the presence of bicarbonate. In this study we test the hypothesis that the increased intracellular pH (pH) caused by bicarbonate stimulates glycolytic activity and the production of lactate by endothelial cells. Primary cultures of bovine corneal endothelial cells (BCEC) were incubated in bicarbonate-free (BF) ringer, a high [HEPES] ringer, and bicarbonate-rich (BR) ringer all at pH 7.

Corneal Endothelial Pump Coupling to Lactic Acid Efflux in the Rabbit and Mouse.

Purpose: Confirm that the corneal endothelial pump uses a lactate-coupled water efflux mechanism.

Methods: Corneal thickness, lactate efflux, and stromal [lactate] were measured in de-epithelialized swollen and nonswollen ex vivo-mounted rabbit corneas perfused with bicarbonate-rich and bicarbonate-free Ringers, ouabain, or acetazolamide to determine if the relationships among these parameters were similar to previous data using intact corneas. The role of barrier function was tested by perfusion with calcium-free EGTA.

Ammonia sensitive SLC4A11 mitochondrial uncoupling reduces glutamine induced oxidative stress.

SLC4A11 is a NH sensitive membrane transporter with H channel-like properties that facilitates Glutamine catabolism in Human and Mouse corneal endothelium (CE). Loss of SLC4A11 activity induces oxidative stress and cell death, resulting in Congenital Hereditary Endothelial Dystrophy (CHED) with corneal edema and vision loss. However, the mechanism by which SLC4A11 prevents ROS production and protects CE is unknown.

Evidence for a GPR18 Role in Chemotaxis, Proliferation, and the Course of Wound Closure in the Cornea.

Purpose: We previously showed that cannabinoid-related GPR18 receptors are present in the murine corneal epithelium, but their function remains unknown. The related CB1 receptors regulate corneal healing, possibly via chemotaxis. We therefore examined a potential role for GPR18 in corneal epithelial chemotaxis and wound healing.

Hypoxia and the Prolyl Hydroxylase Inhibitor FG-4592 Protect Corneal Endothelial Cells From Mechanical and Perioperative Surgical Stress.

Purpose: To determine whether hypoxia preconditioning can protect corneal endothelial cells from mechanical stress and perioperative procedures mimicking Descemet stripping automated endothelial keratoplasty (DSAEK).

Methods: Preconditioning was delivered by 2 hours of 0.5% oxygen incubation in a hypoxia chamber or by exposure to the prolyl hydroxylase inhibitor FG-4592, which prevents hypoxia-inducible factor-1 alpha degradation.

Conditionally Immortal Slc4a11-/- Mouse Corneal Endothelial Cell Line Recapitulates Disrupted Glutaminolysis Seen in Slc4a11-/- Mouse Model.

Purpose: To establish conditionally immortal mouse corneal endothelial cell lines with genetically matched Slc4a11+/+ and Slc4a11-/- mice as a model for investigating pathology and therapies for SLC4A11 associated congenital hereditary endothelial dystrophy (CHED) and Fuchs' endothelial corneal dystrophy.

Methods: We intercrossed H-2Kb-tsA58 mice (Immortomouse) expressing an IFN-γ dependent and temperature-sensitive mutant of the SV40 large T antigen (tsTAg) with Slc4a11+/+ and Slc4a11-/- C57BL/6 mice. The growth characteristics of the cell lines was assessed by doubling time.

Glutaminolysis is Essential for Energy Production and Ion Transport in Human Corneal Endothelium.

Corneal endothelium (CE) is among the most metabolically active tissues in the body. This elevated metabolic rate helps the CE maintain corneal transparency by its ion and fluid transport properties, which when disrupted, leads to visual impairment. Here we demonstrate that glutamine catabolism (glutaminolysis) through TCA cycle generates a large fraction of the ATP needed to maintain CE function, and this glutaminolysis is severely disrupted in cells deficient in NH:H cotransporter Solute Carrier Family 4 Member 11 (SLC4A11).

Fluid transport by the cornea endothelium is dependent on buffering lactic acid efflux.

Maintenance of corneal hydration is dependent on the active transport properties of the corneal endothelium. We tested the hypothesis that lactic acid efflux, facilitated by buffering, is a component of the endothelial fluid pump. Rabbit corneas were perfused with bicarbonate-rich (BR) or bicarbonate-free (BF) Ringer of varying buffering power, while corneal thickness was measured.

Cannabinoid-induced chemotaxis in bovine corneal epithelial cells.

Purpose: Cannabinoid CB1 receptors are found in abundance in the vertebrate eye, with most tissue types expressing this receptor. However, the function of CB1 receptors in corneal epithelial cells (CECs) is poorly understood. Interestingly, the corneas of CB1 knockout mice heal more slowly after injury via a mechanism proposed to involve protein kinase B (Akt) activation, chemokinesis, and cell proliferation.

Human SLC4A11 Is a Novel NH3/H+ Co-transporter.

SLC4A11 has been proposed to be an electrogenic membrane transporter, permeable to Na(+), H(+) (OH(-)), bicarbonate, borate, and NH4 (+). Recent studies indicate, however, that neither bicarbonate or borate is a substrate. Here, we examined potential NH4 (+), Na(+), and H(+) contributions to electrogenic ion transport through SLC4A11 stably expressed in Na(+)/H(+) exchanger-deficient PS120 fibroblasts.

Loss of ion transporters and increased unfolded protein response in Fuchs' dystrophy.

Purpose: Fuchs' endothelial corneal dystrophy (FECD), which affects approximately 5% of the population over 40 in the U.S.A.

CD147 required for corneal endothelial lactate transport.

Purpose: CD147/basigin is a chaperone for lactate:H(+) cotransporters (monocarboxylate transporters) MCT1 and MCT4. We tested the hypothesis that MCT1 and -4 in corneal endothelium contribute to lactate efflux from stroma to anterior chamber and that silencing CD147 expression would cause corneal edema.

Methods: CD147 was silenced via small interfering ribonucleic acid (siRNA) transfection of rabbit corneas ex vivo and anterior chamber lenti-small hairpin RNA (shRNA) pseudovirus in vivo.

Cornea and ocular surface disease: application of cutting-edge optometric research.

: Clinician-scientists bridge the gap between basic research and patient care. At the 2012 Annual Meeting, a symposium highlighting the application of cutting-edge optometric research within the anterior segment was held to present and discuss some of the recent basic scientific advances that will both shape and guide the development of future clinical care practices. This article summarizes this work, bringing together four experts, all clinician-scientists in the field of cornea and ocular surface.

SLC4A11 is an EIPA-sensitive Na(+) permeable pHi regulator.

Slc4a11, a member of the solute linked cotransporter 4 family that is comprised predominantly of bicarbonate transporters, was described as an electrogenic 2Na(+)-B(OH)4(-) (borate) cotransporter and a Na(+)-2OH(-) cotransporter. The goal of the current study was to confirm and/or clarify the function of SLC4A11. In HEK293 cells transfected with SLC4A11 we tested if SLC4A11 is a: 1) Na(+)-HCO3(-) cotransporter, 2) Na(+)-OH(-)(H(+)) transporter, and/or 3) Na(+)-B(OH)4(-) cotransporter.

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⁺.