Effects of all-trans retinoic acid nanoparticles on corneal epithelial wound healing.

BACKGROUND: We have developed inorganically-coated all-trans retinoic acid (atRA) nanoparticles, nano-sized egg-like particles of atRA (NANOEGG®-atRA). The purpose of this study was to determine the effects of NANOEGG®-atRA on corneal wound healing in vivo and in vitro. METHODS: A rabbit corneal epithelial wound healing model was exposed to different concentrations of NANOEGG®-atRA. Wound healing was serially quantified as the ratio of fluorescein-stained area at the selected times to that at baseline. After wound closure, the barrier function of the cornea was determined using low concentrations of tropicamide. At the completion of the experiments, the corneal epithelium was histologically examined. For the in vitro studies, linear scratch wounds were made on cultured SV40-immortalized human corneal epithelial cells (HCE-T). Then, the cells were exposed to different concentrations of NANOEGG®-atRA, and wound healing was determined by the degree of closure of the scratch wound. In addition, the effects of NANOEGG®-atRA on the proliferation of HCE-T cells were determined by WST-8 assays. RESULTS: Exposure to NANOEGG®-atRA decreased the injured area 24 hrs after the ablation. The maximum effect of NANOEGG®-atRA was observed at a concentration of 33 mM. Histologically, no abnormal or differentiated corneal epithelial cells were observed in the histological sections treated with NANOEGG®-atRA. The tropicamide-induced pupillary dilation was significantly slowed in the eyes treated with NANOEGG®-atRA. NANOEGG®-atRA at concentrations of 3.3 and 33 nM induced earlier wound closure in vitro, but did not induce proliferation of HCE-T cells. CONCLUSION: NANOEGG®-atRA promotes wound healing and should be considered for the treatment of wounds of the corneal epithelium.

Diabetes-induced inhibition of voltage-dependent calcium channels in the retinal microvasculature: role of spermine.

PURPOSE: Although decentralized control of blood flow is particularly important in the retina, knowledge of the functional organization of the retinal microvasculature is limited. Here, the authors characterized the distribution and regulation of L-type voltage-dependent calcium channels (VDCCs) within the most decentralized operational complex of the retinal vasculature--the feeder vessel/capillary unit--which consists of a capillary network plus the vessel linking it with a myocyte-encircled arteriole. METHODS: Perforated-patch recordings, calcium-imaging, and time-lapse photography were used to assess VDCC-dependent changes in ionic currents, intracellular calcium, abluminal cell contractility, and lumen diameter, in microvascular complexes freshly isolated from the rat retina. RESULTS: Topographical heterogeneity was found in the distribution of functional VDCCs; VDCC activity was markedly greater in feeder vessels than in capillaries. Experiments showed that this topographical distribution occurs, in large part, because of the inhibition of capillary VDCCs by a mechanism dependent on the endogenous polyamine spermine. An operational consequence of functional VDCCs predominantly located in the feeder vessels is that voltage-driven vasomotor responses are generated chiefly in this portion of the feeder vessel/capillary unit. However, early in the course of diabetes, this ability to generate voltage-driven vasomotor responses becomes profoundly impaired because of the inhibition of feeder vessel VDCCs by a spermine-dependent mechanism. CONCLUSIONS: The regulation of VDCCs by endogenous spermine not only plays a critical role in establishing the physiological organization of the feeder vessel/capillary unit, but also may contribute to dysfunction of this decentralized operational unit in the diabetic retina.

Effect of hypoxia on susceptibility of RGC-5 cells to nitric oxide.

PURPOSE: To determine whether retinal neurons become more susceptible to injury by nitric oxide (NO) under hypoxic conditions. METHODS: Cells from the RGC-5 line were exposed to different concentrations (0.1-100 microM) of S-nitroso-N-acetyl-penicillamine (SNAP), an NO donor, under normoxic and hypoxic (1.0% O(2)) conditions with 5.5 mM glucose or with no glucose. In some experiments, carboxy-PTIO, a scavenger of NO, was added with SNAP. The SNAP-induced cell injury was determined by the WST-8 assay and by the assessment of phosphatidylserine externalization and changes in hypodiploid DNAs. Alterations of mitochondrial membrane potential, superoxide anion formation, cellular adenosine triphosphate (ATP) contents, and caspase activity were also determined after exposure to SNAP. RESULTS: Exposure of RGC-5 cells to SNAP (100 microM) significantly decreased the number of living cells cultured under hypoxic conditions with or without glucose. Coadministration of carboxy-PTIO (1.0 microM) suppressed SNAP-induced cell death. SNAP-induced cell death of cells cultured under hypoxia with glucose was accompanied by increased expression of phosphatidylserine and hypodiploid DNAs. These findings indicated that death was mediated in part by apoptosis. In addition, loss of mitochondrial membrane potential, increase of superoxide formation, and activation of caspase was observed. Cyclosporine A, TEMPOL, and Z-VAD-FMK suppressed cell death. On the other hand, SNAP depleted the ATP contents of cells cultured under hypoxia without glucose, causing mainly necrotic cell death. CONCLUSIONS: These results indicate that RGC-5 cells become susceptible to SNAP under hypoxic conditions in which NO may have greater impact on mitochondrial function.

Effects of chymase on the macular region in monkeys and porcine muller cells: probable involvement of chymase in the onset of idiopathic macular holes.

OBJECTIVES: To investigate chymase involvement in idiopathic macular hole onset, the effects of chymase on monkey eyes and cultured Muller cells were investigated. METHODS: Immunohistochemistry using antinestin and antiglial fibrillary acidic protein antibodies was performed in a normal monkey eye. After chymase was injected into the monkey vitreous, histological changes in the retina were evaluated using the TdT-mediated dUTP nick-end labeling (TUNEL) assay. Expression of c-kit, a stem cell factor receptor, and nestin was examined in porcine Muller cells cultured with basic fibroblast growth factor. The effects of chymase on proliferation and TUNEL staining in Muller cells were also examined. RESULTS: The number of nestin and glial fibrillary acidic protein-positive cells was higher in the macula than in other regions. Thickening of the posterior hyaloid membrane and some apoptotic cells were found in the macula of chymase-treated eyes. The expression of c-kit and nestin in Muller cells was shown and enhanced when cultured with basic fibroblast growth factor. Exposure to chymase inhibited Muller cell proliferation and produced TUNEL-positive cells. CONCLUSIONS: There might be Muller cells possessing atypical properties near the macular region and chymase might cause fibrosis and apoptosis through these cells. These findings suggest that increased chymase activity may result in idiopathic macular hole onset.

Extracellular lactate as a dynamic vasoactive signal in the rat retinal microvasculature.

We tested the hypothesis that extracellular lactate regulates the function of pericyte-containing retinal microvessels. Although abluminally positioned pericytes appear to adjust capillary perfusion by contracting and relaxing, knowledge of the molecular signals that regulate the contractility of these mural cells is limited. Here, we focused on lactate because this metabolic product is in the retinal extracellular space under both physiological and pathophysiological conditions. In microvessels freshly isolated from the adult rat retina, we used perforated-patch pipettes to monitor ionic currents, fura-2 to measure calcium levels, and time-lapse photography to visualize changes in mural cell contractility and lumen diameter. During lactate exposure, pericyte calcium rose; these cells contracted, and lumens constricted. This contractile response appears to involve a cascade of events resulting in the inhibition of Na+/Ca2+ exchangers (NCXs), the decreased of which function causes pericyte calcium to increase and contraction to be triggered. On the basis of our observation that gap junction uncouplers minimized the lactate-induced rise in pericyte calcium, we propose that the NCXs inhibited by lactate are predominately located in the endothelium. Indicative of the importance of endothelial/pericyte gap junctions, uncouplers of these junctions switched the pericyte response to lactate from contraction to relaxation. In addition, we observed that hypoxia, which closes microvascular gap junctions, also switched lactate's effect from vasocontraction to vasorelaxation. Thus the response of pericyte-containing retinal microvessels to extracellular lactate is metabolically modulated. The ability of lactate to serve as a vasoconstrictor when energy supplies are ample and a vasodilator under hypoxic conditions may be an efficient mechanism to link capillary function with local metabolic need.

Endothelin-1 enhances glutamate-induced retinal cell death, possibly through ETA receptors.

PURPOSE: To determine the modification of the glutamate-induced death of retinal neurons by endothelin (ET)-1. METHODS: Cultured retinal neurons from fetal rats were exposed to glutamate (1.0 mM) alone or glutamate with ET-1 (10(-10)-10(-7)M) for 10 minutes. Neuronal death was assessed by the trypan blue exclusion or TUNEL assays at 2, 6, and 24 hours after the exposure. The effects of adding BQ-123 or BQ-788, ET(A), and ET(B) receptor antagonists, respectively, in combination with ET-1 was also assessed. RESULTS: Immunohistochemical analyses showed that the ETs as well as ET(A) and ET(B) receptors were expressed on cultured retinal neurons consisting mainly of amacrine cells. A brief exposure of the cultured retinal neurons to glutamate alone significantly increased the number of dead cells, and the addition of ET-1 with glutamate caused a further significant increase in retinal neuronal death compared with the cells exposed to glutamate alone. A significant increase in neuronal death was detected at doses of 10 nM of ET-1 and higher after a 24-hour exposure (P < 0.05, Dunnett), whereas brief exposure of neurons to up to 1 microM ET-1 alone did not cause delayed cell death of neurons. BQ-123 (10 nM) suppressed the enhancement of retinal toxicity caused by ET-1 (10 nM), whereas BQ-788 had no significant effect. CONCLUSIONS: These results indicate that ET-1 enhances glutamate-induced retinal cell death, possibly through ET(A) receptors. ET-1 may act synergistically with glutamate to damage retinal neurons under hypoxic conditions.

Regulation of P2X7-induced pore formation and cell death in pericyte-containing retinal microvessels.

The purpose if this study was to elucidate how extracellular ATP causes cell death in the retinal microvasculature. Although ATP appears to serve as a vasoactive signal acting via P2X(7) and P2Y(4) purinoceptors, this nucleotide can kill microvascular cells of the retina. Because P2X(7) receptor activation causes transmembrane pores to form and microvascular cells to die, we initially surmised that pore formation accounted for ATP's lethality. To test this hypothesis, we isolated pericyte-containing microvessels from rat retinas, assessed cell viability using Trypan blue dye exclusion, detected pores by determining the uptake of the fluorescent dye YO-PRO-1, measured intracellular Ca(2+) with the use of fura-2, and monitored ionic currents via perforated patch pipettes. As predicted, ATP-induced cell death required P2X(7) receptor activation. However, we found that pore formation was minimal because ATP's activation of P2Y(4) receptors prevented P2X(7) pores from forming. Rather than opening lethal pores, ATP kills via a mechanism involving voltage-dependent Ca(2+) channels (VDCC). Our experiments suggest that when high concentrations of ATP caused nearly all microvascular P2X(7) receptor channels to open, the resulting profound depolarization opened VDCC. Consistent with lethal Ca(2+) influx via VDCC, ATP-induced cell death was markedly diminished by the VDCC blocker nifedipine or a nitric oxide (NO) donor that inhibited microvascular VDCC. We propose that purinergic vasotoxicity is normally prevented in the retina by NO-mediated inhibition of VDCC and P2Y(4)-mediated inhibition of P2X(7) pore formation. Conversely, dysfunction of these protective mechanisms may be a previously unrecognized cause of cell death within the retinal microvasculature.

Effects of angiotensin II on the pericyte-containing microvasculature of the rat retina.

The aim of this study was to identify the mechanisms by which angiotensin II alters the physiology of the pericyte-containing microvasculature of the retina. Despite evidence that this vasoactive signal regulates capillary perfusion by inducing abluminal pericytes to contract and thereby microvascular lumens to constrict, little is known about the events linking angiotensin exposure with pericyte contraction. Here, using microvessels freshly isolated from the adult rat retina, we monitored pericyte currents via perforated-patch pipettes, measured pericyte calcium levels with fura-2 and visualized pericyte contractions and lumen constrictions by time-lapse photography. We found that angiotensin activates nonspecific cation (NSC) and calcium-activated chloride channels; the opening of these channels induces a depolarization that is sufficient to activate the voltage-dependent calcium channels (VDCCs) expressed in the retinal microvasculature. Associated with these changes in ion channel activity, intracellular calcium levels rise, pericytes contract and microvascular lumens narrow. Our experiments revealed that an influx of calcium through the NSC channels is an essential step linking the activation of AT(1) angiotensin receptors with pericyte contraction. Although not required in order for angiotensin to induce pericytes to contract, calcium entry via VDCCs serves to enhance the contractile response of these cells. In addition to activating nonspecific cation, calcium-activated chloride and voltage-dependent calcium channels, angiotensin II also causes the functional uncoupling of pericytes from their microvascular neighbours. This inhibition of gap junction-mediated intercellular communication suggests a previously unappreciated complexity in the spatiotemporal dynamics of the microvascular response to angiotensin II.

ATP: a vasoactive signal in the pericyte-containing microvasculature of the rat retina.

In this study we tested the hypothesis that extracellular ATP regulates the function of the pericyte-containing retinal microvessels. Pericytes, which are more numerous in the retina than in any other tissue, are abluminally located cells that may adjust capillary perfusion by contracting and relaxing. At present, knowledge of the vasoactive molecules that regulate pericyte function is limited. Here, we focused on the actions of extracellular ATP because this nucleotide is a putative glial-to-vascular signal, as well as being a substance released by activated platelets and injured cells. In microvessels freshly isolated from the adult rat retina, we monitored ionic currents via perforated-patch pipettes, measured intracellular calcium levels with the use of fura-2, and visualized microvascular contractions with the aid of time-lapse photography. We found that ATP induced depolarizing changes in the ionic currents, increased calcium levels and caused pericytes to contract. P2X7 receptors and UTP-activated receptors mediated these effects. Consistent with ATP serving as a vasoconstrictor for the pericyte-containing microvasculature of the retina, the microvascular lumen narrowed when an adjacent pericyte contracted. In addition, the sustained activation of P2X7 receptors inhibited cell-to-cell electrotonic transmission within the microvascular networks. Thus, ATP not only affects the contractility of individual pericytes, but also appears to regulate the spatial and temporal dynamics of the vasomotor response.