Hydrogen sulfide is synthesized endogenously in both retinal artery and retina mostly via CSE.

Hydrogen sulfide (H2S) is an important gasotransmitter expressed in various tissues of the organism, including the eye. It is known that H2S is localized especially in the retina and corneal layers in bovine eye. The enzymes that mediate H2S synthesis are 3-mercaptopyruvate sulfurtransferase (3-MST), cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE). Herein, we aimed to investigate the concentration levels and distribution profiles of these enzymes in bovine retina and retinal artery. Enzyme levels were measured by ELISA and distribution were determined by immunofluorescence microscopic analysis. Much higher concentrations of CBS and CSE have been detected in the retinal artery compared to the retina. In both tissues, particulary 3-MST was found at the lowest level while, CSE was determined to be the most abundant enzyme among the others. CBS distribution was shown in both endothelial and smooth muscle layers, while CSE was seen especially in the endothelial layer of the retinal artery. In the retina, CBS and CSE were expressed in cone-basil cells and retinal ganglion cells, while CSE was also present in bipolar cells. Our results indicated that H2S is synthesized endogenously in ocular tissues. The widespread expression of H2S synthesizing enzymes in the retina and retinal artery of the bovine eye, which has anatomical similarities with the human eye, may suggest a protective role for H2S against retinal vascular diseases as well as a regulatory role in the retinal vascular tone.

COX-2 inhibition improves retinal function in rats' ischemic eyes.

PURPOSE: Retinal ischemia is a relatively simple model for studies in pharmacological neuroprotective intervention. The role of cyclooxygenase (COX) enzymes in ischemic insult has been variously shown to either increase or decrease ischemic damage. The purpose of this study was to assess the role of COX-1 and COX-2 in rat retinal ischemic functional damage. METHODS: Ischemia was achieved by elevating intraocular pressure for 60 min. White flash electroretinogram (ERG) was recorded by contact lens electrodes containing an integral light emitting diode source. ERG was recorded on post-ischemia (PI) days-1 (baseline), 1, 3, and 7. The b-wave amplitude, b-wave implicit time, and oscillatory potentials (OPs) were analyzed. The expression of COX-2 and HSP70i was assessed by Western analysis on day 1 PI. RESULTS: Ischemia caused attenuation of OPs, a decrease in the b-wave amplitude, and an increase in b-wave implicit time, accompanied by the increased expression of COX-2 and HSP70i proteins. Selective COX-2 inhibition markedly increased b-wave amplitude and enhanced retinal HSP70i induction, whereas COX-1 or nonselective and irreversible inhibition of both COX isoenzymes did not affect the retinal function or the expression of these proteins. High-dose aspirin prevented partial recovery from ischemic damage. Administration of a synthetic PGF2α analog, or a lipoxygenase inhibitor, had little effect on ischemic damage, but affected nonischemic ERG. CONCLUSIONS: COX-2 appears to mediate some of the ischemic retinal functional damage, possibly by inhibiting the induction of HSP-70i. We propose that selective COX-2 inhibitors may be useful in pathological conditions involving ischemic retinal insult.

Mutation of IGFBP7 causes upregulation of BRAF/MEK/ERK pathway and familial retinal arterial macroaneurysms.

Insulin-like growth factor binding proteins (IGFBPs) play important physiological functions through the modulation of IGF signaling as well as IGF-independent mechanisms. Despite the established role of IGFs in development, a similar role for the seven known IGFBPs has not been established in humans. Here, we show that an autosomal-recessive syndrome that consists of progressive retinal arterial macroaneurysms and supravalvular pulmonic stenosis is caused by mutation of IGFBP7. Consistent with the recently established inhibitory role of IGFBP7 on BRAF signaling, the BRAF/MEK/ERK pathway is upregulated in these patients, which may explain why the cardiac phenotype overlaps with other disorders characterized by germline mutations in this pathway. The retinal phenotype appears to be mediated by a role in vascular endothelium, where IGFBP7 is highly expressed.

Comparative study of the retinal vessel anatomy of rhesus monkeys and humans.

OBJECTIVES: This study aims to compare retinal vessel anatomy of normal rhesus monkeys and humans and to provide a basis from a structural perspective for the use of rhesus monkey as an experimental model in future studies of retinal vessels. METHODS: The retinas of six normal rhesus monkey eyes and eight human eyecups following corneal transplantation were obtained and stained using adenosine diphosphatase methods. The distributions, orders, layers of the retinal vessels and the perifoveal vascular ring were compared. RESULTS: With adenosine diphosphatase staining, distinct retinal vessels were fully discernable from the first order surrounding the optic disc to the fifth order. There were no statistically significant differences between rhesus monkeys and humans in the percentages of vessels surrounding the optic disc at the equator and the peripheral region. Vascular networks in both species were arranged in several layers around the optic disc, two anastomotic layers at the equator and one sparse layer peripherally. Capillaries at the macular area were quite dense and an intact perifoveal vascular ring was observed. No differences were observed between rhesus monkeys and humans in the percentage area of the vessels and the area, perimeter and diameter of the perifoveal vascular ring. CONCLUSIONS: The distributions, orders, layers and the perifoveal vascular ring of the retinal vessels of rhesus monkey are quite similar to those of humans. The data suggest that from an anatomical perspective, the rhesus monkey is a good animal model for the study of human retinal vessels, particularly the macular capillaries.

Mitogen-activated protein kinases in the porcine retinal arteries and neuroretina following retinal ischemia-reperfusion.

PURPOSE: The aim of the present study was to examine changes in the expression of intracellular signal-transduction pathways, specifically mitogen-activated protein kinases, following retinal ischemia-reperfusion. METHODS: Retinal ischemia was induced by elevating the intraocular pressure in porcine eyes, followed by 5, 12, or 20 h of reperfusion. The results were compared to those of the sham- operated fellow eye. The retinal arteries and neuroretina were isolated separately and examined. Tissue morphology and DNA fragmentation were studied using histology. Extracellular signal-regulated kinase 1 and 2 (ERK1/2), p38, c-junNH(2)-terminal kinases (JNK), and c-jun protein and mRNA expression were examined using immunofluorescence staining, western blot, and real-time PCR techniques. RESULTS: Pyknotic cell nuclei, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells, and glial fibrillary acidic protein mRNA expression were increased in ischemia, suggesting injury. Phosphorylated ERK1/2 protein levels were increased in the neuroretina following ischemia, while mRNA levels were unaltered. p38 protein and mRNA levels were not affected by ischemia. Immunofluorescence staining for phosphorylated p38 was especially intense in the retinal blood vessels, while only weak in the neuroretina. Phosphorylated JNK protein and mRNA were slightly decreased in ischemia. Phosphorylated c-jun protein and mRNA levels were higher in the neuroretina after ischemia-reperfusion. CONCLUSIONS: Retinal ischemia-reperfusion alters expression of mitogen-activated protein kinases, particularly ERK1/2, in the neuroretina and retinal arteries. The development of pharmacological treatment targeting these intracellular transduction pathways may prevent injury to the eye following retinal circulatory failure.

Protein kinase C in porcine retinal arteries and neuroretina following retinal ischemia-reperfusion.

PURPOSE: Identification of the intracellular signal-transduction pathways activated in retinal ischemia may be important in revealing novel pharmacological targets. To date, most studies have focused on identifying neuroprotective agents. The retinal blood vessels are key organs in circulatory failure, and this study was therefore designed to examine the retinal vasculature separately from the neuroretina. METHODS: Retinal ischemia was induced by elevating the intraocular pressure in porcine eyes, followed by 5, 12, or 20 h of reperfusion. Protein kinase C (PKC)alpha, PKCbeta1, and PKCbeta2 mRNA levels, and protein expression were determined using real-time PCR, western blot, and immunofluorescence staining techniques. RESULTS: The retinal arteries could easily be dissected free and studied separately from the neuroretina in this porcine model. The PKCalpha, PKCbeta1, and PKCbeta2 mRNA levels tended to be lower in ischemia-reperfused than in sham-operated eyes in both the retinal arteries and the neuroretina. This was most prominent after 5 h, and less pronounced after 12 h and 20 h of reperfusion. Likewise, the protein levels of PKCalpha, PKCbeta1, and PKCbeta2 were slightly lower following ischemia-reperfusion when compared to sham-operated eyes. PKCalpha, PKCbeta1, and PKCbeta2 immunostaining were observed in bipolar cells of the neuroretina and in endothelial cells, and to a low extent in the smooth muscle layer, of the retinal arteries. CONCLUSIONS: Retinal ischemia followed by reperfusion results in lower levels of PKC in both the neuroretina and retinal arteries. New targets for pharmacological treatment may be found by studying the retinal vasculature so as to identify the intracellular signal-transduction pathways involved in the development of injury following retinal circulatory failure.

Amyloid beta(1-42) and its beta(25-35) fragment induce activation and membrane translocation of cytosolic phospholipase A2 in bovine retina capillary pericytes.

We investigated changes in cytosolic phospholipase A(2) (cPLA(2)) and calcium-independent PLA(2) (iPLA(2)) activities in bovine retina capillary pericytes after stimulation with 50 microM amyloid-beta (Abeta) (1-42) and its (25-35) fragment, over 24 h (mild, sublethal model of cell damage). In the presence of Abeta peptides, we found that cPLA(2) activity was increased and translocated from the cytosolic fraction to the membrane system, particularly in the nuclear region. Reversed-sequence Abeta(35-25) peptide did not stimulate or induce cPLA(2) translocation. Exposure to both Abeta peptides had no significant effect on cPLA(2) protein content as tested by Western immunoblot analysis. The addition of Abetas to quiescent pericytes was followed by phosphorylation of cPLA(2) and arachidonic acid release. Treatment with inhibitors (AACOCF(3), staurosporine and cycloheximide) resulted in a sharp decrease in basal and stimulated cPLA(2) activity. Inactivating effects of bromoenol lactone (BEL), inhibitor of iPLA(2), demonstrated that the stimulation of total PLA(2) activity by Abetas was mediated by both PLA(2) enzymes. Taken together with our previous observations that both Abeta peptides may induce hydrolysis of phosphatidylcholine, the present results provide evidence that this process is cooperatively mediated by cPLA(2) activation/translocation and iPLA(2) activation. The effect is very likely triggered by a mild prooxidant mechanism which was not able to divert the cell to degeneration. The data confirm the hypothesis that pericytes could be a target of potential vascular damage and reactivity during processes involving amyloid accumulation.

Retinopathy in diabetic (KKA gamma) mice: diabetic microvascular changes to the retina in KKA gamma mice revealed by light and electron microscopy.

Pericytic changes in the retinal vessels of diabetic (KKA gamma) and control (C57BL) mice were studied by light and electron microscopy. An improved histochemical technique for alkaline phosphatase was used in the light microscopic study. In the control mice, a continuous pathway was identified extending from the retinal arterioles, via the superficial and deep retinal capillaries, to the retinal venules. The deep retinal capillaries formed networks and were localized within the deeper retinal layers; the retinal arterioles, superficial capillaries, and venules were present in the nerve fiber layer. Examination of KKA gamma mice, aged 16 to 28 weeks, revealed engorgement of the arterioles, hypertrophy of the pericytes (which contained numerous actin filaments) within the superficial retinal capillaries, and narrowing of the deep retinal capillaries. These microvascular changes indicate retinal hyperperfusion, local hypertension of the superficial retinal capillaries, adaptive hyperfunctional changes in the pericytes of these capillaries, and ischemia of the deep retinal capillaries. The pericytic changes observed in the diabetic capillaries contrasted sharply with previous reports; an explanation for this variance is suggested.