Application of Rho Kinase Inhibitors for the Treatment of Corneal Endothelial Diseases

ROCK (Rho kinase) signaling regulates a wide spectrum of fundamental cellular events and is involved in a variety of pathological conditions. It has therefore attracted research interest as a potential therapeutic target for combating various diseases. We showed that inhibition of ROCK enhances cell proliferation, promotes cell adhesion onto a substrate, and suppresses apoptosis of corneal endothelial cells (CECs). In addition, we reported that a ROCK inhibitor enhances wound healing in the corneal endothelium in animal models and in pilot clinical research. We also demonstrated the usefulness of a ROCK inhibitor as an adjunct drug in tissue engineering therapy as it enhances the engraftment of CECs onto recipient corneas. In 2013, we initiated a clinical trial to test the effectiveness of injection of cultured human CECs into the anterior chamber of patients with corneal endothelial decompensation. This paper reviews the accumulating evidence supporting the potency of ROCK inhibitors in clinical use, both as eye drops and as adjunct drugs in cell-based therapies, for the treatment of corneal endothelial decompensation

Development of the Water Resistojet Propulsion System for Deep Space Exploration by the CubeSat: EQUULEUS

In this study, Water micro-propulsion system AQUARIUS (AQUA ResIstojet propUlsion System) is proposed for 6U CubeSat: EQUULEUS to explore the deep space. AQUARIUS uses storable, safe and non-toxic propellant: water, which allows for downsizing of whole propulsion system to 2U and storing 1.2 kg water. Liquid propellant storage allows design of all propulsion systems below 100 kPa. The waste heat of communication components is reused to cover high latent heat of water. AQUARIUS has 4.0 mN and specific impulse of 70 s by less than 20 W power consumption. Breadboard model was designed and tested successfully. Engineering model is under developments and operations by using whole systems of EQUULEUS. AQUARIUS will be equipped with EQUULEUS scheduled to be launched in 2019 by SLS (Space Launch System)

Rho-associated kinase inhibitor eye drop (Ripasudil) transiently alters the morphology of corneal endothelial cells

PURPOSE: Ripasudil (Glanatec), a selective Rho-associated coiled coil-containing protein kinase (ROCK) inhibitor, was approved in Japan in September 2014 for the treatment of glaucoma and ocular hypertension. The purpose of this study was to investigate the effect of ripasudil eye drops on corneal endothelial morphology, as ROCK signaling is known to modulate the actin cytoskeleton. METHODS: Morphological changes in the corneal endothelium were evaluated in human subjects by specular and slit-lamp microscopy, following topical administration of ripasudil. We also used a rabbit model to evaluate the effect of ripasudil on clinical parameters of the corneal endothelium. Twenty-four hours after ripasudil application, corneal specimens were evaluated by phalloidin staining, immunohistochemical analysis, and electron microscopy. RESULTS: Specular microscopy revealed morphological changes in human eyes, and slit-lamp microscopy showed guttae-like findings. The rabbit model showed morphological changes similar to those seen in human eyes after ripasudil administration. Electron microscopy demonstrated that these alterations are due to the formation of protrusions along the cell-cell borders, but this formation is transient. Expression of corneal endothelial function-related markers was not disrupted; corneal thickness and corneal volume were not changed; and no cell death was observed following ripasudil administration. CONCLUSIONS: Ripasudil induces transient guttae-like findings in humans, most likely due to protrusion formation along intracellular borders caused by the reduction in actomyosin contractility of the corneal endothelial cells. No severe adverse effects were observed. Physicians should be aware that ROCK inhibitors can cause these guttae-like findings, to avoid misdiagnosing patients as having Fuchs endothelial corneal dystrophy. (www.umin.ac.jp/ctr number, UMIN000018340.)

Development of Poly Lactic/Glycolic Acid (PLGA) Microspheres for Controlled Release of Rho-Associated Kinase Inhibitor

Purpose. The purpose of this study was to investigate the feasibility of poly lactic/glycolic acid (PLGA) as a drug delivery carrier of Rho kinase (ROCK) inhibitor for the treatment of corneal endothelial disease. Method. ROCK inhibitor Y-27632 and PLGA were dissolved in water with or without gelatin (W1), and a double emulsion [(W1/O)/W2] was formed with dichloromethane (O) and polyvinyl alcohol (W2). Drug release curve was obtained by evaluating the released Y-27632 by using high performance liquid chromatography. PLGA was injected into the anterior chamber or subconjunctiva in rabbit eyes, and ocular complication was evaluated by slitlamp microscope and histological analysis. Results. Y-27632 incorporated PLGA microspheres with different molecular weights, and different composition ratios of lactic acid and glycolic acid were fabricated. A high molecular weight and low content of glycolic acid produced a slower and longer release. The Y-27632 released from PLGA microspheres significantly promoted the cell proliferation of cultured corneal endothelial cells. The injection of PLGA did not induce any evident eye complication. Conclusions. ROCK inhibitor-incorporated PLGA microspheres were fabricated, and the microspheres achieved the sustained release of ROCK inhibitor over 7–10 days in vitro. Our data should encourage researchers to use PLGA microspheres for treating corneal endothelial diseases

Cell surface markers of functional phenotypic corneal endothelial cells. Invest Ophthalmol Vis Sci 2014

PURPOSE. Cultured human corneal endothelial cells (HCECs) are anticipated to serve as an alternative to donor corneas for the treatment of corneal endothelial dysfunction. However, corneal endothelial cells (CECs) tend to exhibit fibroblastic transformation, thereby losing their functional phenotype when cultured. The purpose of this study was to investigate the usefulness of surface markers of CECs displaying fibroblastic phenotypes as a means of cell characterization. METHODS. The expression levels of 242 cell surface antigens were screened in cultured human and monkey CECs using flow cytometry. An expression intensity ratio of nonfibroblastic/ fibroblastic CECs > 2 and of fibroblastic/nonfibroblastic CECs > 2 were selected as indicating nonfibroblastic and fibroblastic markers, respectively. Nonfibroblastic and fibroblastic CECs were mixed, and CD73-positive and -negative cells were sorted using flow cytometry and further cultured. The functional phenotype of the sorted cells was evaluated according to morphology and the expression of function-related (Na þ /K þ -ATPase and ZO-1) and fibroblastic (type I collagen and fibronectin) markers. RESULTS. Flow cytometry analysis demonstrated that CD98, CD166, and CD340 are elevated in HCECs of nonfibroblastic phenotype, while CD9, CD49e, CD44, and CD73 are markers of fibroblastic phenotype HCECs. The CECs that sorted as CD73-negative exhibited normal hexagonal morphology and expressed functional markers, whereas CECs that sorted as CD73-positive exhibited the fibroblastic phenotype. CONCLUSIONS. These markers will be useful for quality control to characterize the phenotype of cells destined for tissue engineering-based therapy. In addition, this selection protocol will provide a novel method for purification of functional cells

A novel superior factor widely controlling the rice grain quality

Synthesis of storage starch and protein accumulation is the main action of endosperm organogenesis in term of the economic importance of rice. This event is strongly disturbed by abiotic stresses such as high temperature; thus, the upcoming global warming will cause a crisis with a great impact on food production^1,2^. The enzymes for the protein storage and starch synthesis pathway should work in concert to carry out the organogenesis of rice endosperm^3-5^, but the regulatory mechanism is largely unknown. Here we show that a novel regulatory factor, named OsCEO1, acts as the conductor of endosperm organogenesis during the rice grain filling stage. The physiological properties of _floury-endosperm-2_ (_flo2_) mutants showed many similarities to symptoms of grains developed under high-temperature conditions, suggesting important roles of the responsible gene in sensitivity to high-temperature stress. Our map-based cloning identified the responsible gene for the _flo2_ mutant, _OsCEO1_, which has no homology to any genes of known function. The _OsCEO1_ belongs to a novel conserved gene family and encodes a protein composed of 1,720 amino acid residues containing a TPR (tetratricopeptide repeat) motif, which is considered to mediate a protein-protein interaction. The yeast two-hybrid analysis raised an unknown protein showing homology to a late embryogenesis abundant protein and a putative basic helix-loop-helix protein as candidates for the direct interactor for _OsCEO1_, whereas no enzyme genes for the synthesis of storage substances were detected. The _flo2_ mutant exhibited reduced expression of several genes for putative regulatory proteins as well as many enzymes involved in storage starch and proteins. These results suggest that _OsCEO1_ is a superior conductor of the novel regulatory cascade of endosperm organogenesis and may have important roles in the response to high-temperature stress

Pressure-induced structural phase transition and new superconducting phase in UTe2

We report on the crystal structure and electronic properties of the heavy fermion superconductor UTe2 at high pressure up to 11 GPa, as investigated by X-ray diffraction and electrical resistivity experiments. The X-ray diffraction measurements under high pressure using a synchrotron light source reveal anisotropic linear compressibility of the unit cell up to 3.5 GPa, while a pressure-induced structural phase transition is observed above 3.5-4GPa at room temperature, where the body-centered orthorhombic crystal structure with the space group Immm changes into a body-centered tetragonal structure with the space group I4/mmm. The molar volume drops abruptly at the critical pressure, while the distance between the first-nearest neighbor of U atoms increases, implying a switch from the heavy electronic states to the weakly correlated electronic states. Surprisingly, a new superconducting phase at pressures higher than 7 GPa was detected at Tsc above 2K with a relatively low upper-critical field, Hc2(0). The resistivity above 3.5GPa, thus, in the high-pressure tetragonal phase, shows a large drop below 230 K, which may also be related to a considerable change from the heavy electronic states to the weakly correlated electronic states.Comment: 11 pages, 9 figure

Sustained activation of the unfolded protein response induces cell death in Fuchs' endothelial corneal dystrophy

Purpose: The unfolded protein response (UPR) is believed to play a role in the pathogenesis of Fuchs' endothelial corneal dystrophy (FECD). The purpose of this study was to investigate whether unfolded proteins accumulate in the corneal endothelium in FECD and if they are involved in triggering cell death. Methods: Descemet's membranes with corneal endothelial cells (CECs) were obtained during keratoplasty, and expression of aggresomes, type 1 collagen, fibronectin, and agrin was evaluated. Endoplasmic reticulum (ER) stress of immortalized human CECs from non-FECD subjects and from FECD patients (iHCEC and iFECD, respectively) were evaluated. The effect of MG132-mediated aggresome formation on the UPR and intrinsic pathway and the effect of mitochondrial damage on UPR were also examined. The effect of CHOP knockdown on the ER stress–mediated intrinsic pathway was also evaluated. Results: Aggresome formation was higher in iFECD than in iHCEC and was colocalized with type 1 collagen, fibronectin, and agrin. GRP78, phosphorylated IRE1, PERK, and CHOP showed higher activation in iFECD than in iHCEC. MG132-mediated aggresome formation upregulated ER stress sensors, the mitochondrial membrane potential drop, cytochrome c release to the cytoplasm, and activation of caspase-9 and -3. By contrast, staurosporine-mediated mitochondrial damage did not induce ER stress. Knockdown of CHOP attenuated the ER stress-induced cleavage of caspase-9, which is caused by intrinsic pathway activation. Conclusions: Excessive synthesis of extracellular matrix proteins induced unfolded protein accumulation in FECD. Prolonged ER stress–mediated cell death, occurring via the intrinsic apoptotic signaling pathway, therefore might be associated with the pathogenesis of FECD

A Surgical Cryoprobe for Targeted Transcorneal Freezing and Endothelial Cell Removal

PURPOSE: To examine the effects of transcorneal freezing using a new cryoprobe designed for corneal endothelial surgery. METHODS: A freezing console employing nitrous oxide as a cryogen was used to cool a series of different cryoprobe tip designs made of silver for high thermal conductivity. In vitro studies were conducted on 426 porcine corneas, followed by preliminary in vivo investigations on three rabbit corneas. RESULTS: The corneal epithelium was destroyed by transcorneal freezing, as expected; however, the epithelial basement membrane remained intact. Reproducible endothelial damage was optimally achieved using a 3.4 mm diameter cryoprobe with a concave tip profile. Stromal edema was seen in the pre-Descemet's area 24 hrs postfreeze injury, but this had been resolved by 10 days postfreeze. A normal collagen fibril structure was seen 1 month postfreeze, concurrent with endothelial cell repopulation. CONCLUSIONS: Transcorneal freezing induces transient posterior stromal edema and some residual deep stromal haze but leaves the epithelial basement membrane intact, which is likely to be important for corneal re-epithelialization. Localized destruction of the endothelial monolayer was achieved in a consistent manner with a 3.4 mm diameter/concave profile cryoprobe and represents a potentially useful approach to remove dysfunctional corneal endothelial cells from corneas with endothelial dysfunction