Autocrine and Paracrine Secretion of Vascular Endothelial Growth Factor in the Pre-Hypoxic Diabetic Retina.

Vascular endothelial growth factor (VEGF) is well established as the main agent responsible for vascular leakage and angiogenesis in the diabetic retina. While VEGF can have positive effects on hyperglycemia stressed retinal tissues, it also plays a role in events progressing to the oxygen- stressed, i.e. hypoxic, diabetic retina. Some VEGF makes its way to the retina from systemic sources and some is produced locally within the eye. Hyperglycemia, oxidants, inflammation, and advanced glycation end-products are all stimulants to VEGF production, both in the hypoxic and the pre-hypoxic retina. Endothelial cells, pericytes, Müller cells, microglia, astrocytes, retinal pigment epithelium and neurons have all been known to produce VEGF at some point in retinal development or in disease. Excessive VEGF production in the early diabetic retina can lead to retinal exposure or mechanisms which exacerbate further damage. While Müller cells are likely the most significant producer of VEGF in the pre-hypoxic retina, other VEGF producing cells may also play a role due to their proximity to vessels or neurons. Study of the release of VEGF by retinal cells in hyperglycemia conditions, may help identify targets for early treatment and prevent the serious consequences of diabetic retinopathy.

The role of microglia in diabetic retinopathy.

There is growing evidence that chronic inflammation plays a role in both the development and progression of diabetic retinopathy. There is also evidence that molecules produced as a result of hyperglycemia can activate microglia. However the exact contribution of microglia, the resident immune cells of the central nervous system, to retinal tissue damage during diabetes remains unclear. Current data suggest that dysregulated microglial responses are linked to their deleterious effects in several neurological diseases associated with chronic inflammation. As inflammatory cytokines and hyperglycemia disseminate through the diabetic retina, microglia can change to an activated state, increase in number, translocate through the retina, and themselves become the producers of inflammatory and apoptotic molecules or alternatively exert anti-inflammatory effects. In addition, microglial genetic variations may account for some of the individual differences commonly seen in patient's susceptibility to diabetic retinopathy.

Effects of tamoxifen versus raloxifene on retinal capillary endothelial cell proliferation.

PURPOSE: Endothelial cell proliferation in angiogenesis is active in conditions such as cancers and diabetic retinopathy. Tamoxifen (T) and raloxifene (R) have been compared in numerous studies as a prophylaxis for breast cancer, and T is used to treat breast cancer. T, unlike R, has been linked to an increase in uterine cancers, thrombo-embolic events, and cataract. The purpose of our study was to evaluate the efficacies of T and R in reducing estrogen-induced retinal capillary endothelial cell proliferation. METHODS: Rhesus monkey retinal capillary endothelial cells (ATCC RF/6A) were used to assay cell proliferation when treated with 0.0, 0.1, 1.0, and 10.0 nM 17 ß estradiol (E2) for 24 and 48 h. Viable cells were counted using a Neubauer hemocytometer with a trypan blue exclusion method to determine the number of viable cells. Cell counts were also performed using 1.0 nM E2 with 0.01, 0.1, 1.0, and 10.0 nM concentrations of either T or R. Cell medium, collected at 24 h, was evaluated for vascular endothelial growth factor and pigment epithelium-derived factor. RESULTS: Viable cells were significantly greater in cultures treated with 1.0 or 10.0 nM E2, compared to cells treated with 0.0 or 0.1 nM E2 both at 24 and 48 h. Viable cell counts were reduced significantly in cultures treated with 0.1, 1.0, or 10.0 nM T or R in addition to the 1.0 nM E2. Cell counts were not significantly different when comparing equal concentrations of T and R, that is, 1.0 nM E2+1 nM T or R. Vascular endothelial growth factor and pigment epithelium-derived factor protein/10,000 cells was reduced by 1.0 nM E2, but returned to higher levels with the introduction of T and R to growth media. CONCLUSIONS: T and R showed similar potency in inhibiting estrogen-induced retinal capillary endothelial cell proliferation. Considering drug safety profiles, our results, when extended to animals and humans, suggest that R is preferable to T in treating angiogenic retinal diseases. Further studies on the signaling mechanism of estrogen-induced endothelial cell proliferation may lead to new treatment strategies in the treatment of ocular angiogenic diseases.

Ginsenoside-Rb1 Induces ARPE-19 Proliferation and Reduces VEGF Release.

Rb1, a ginsenoside from ginseng root extract, possesses antiangiogenic effects, but its role on ocular cells has not been studied. We hypothesize that Rb1 inhibits the production of the angiogenic cytokine VEGF from ARPE-19 cells, leading to a significant reduction in the proliferation of ocular vasculatures. Data from our experiments show that Rb1 induced an increase in the number of ARPE cells in culture, while VEGF release (pg/10,000 viable cells) was significantly reduced. Treatment with VEGF and cotreatment with Rb1 and VEGF showed that this Rb1-induced cell proliferation was mediated by VEGF. Because VEGF from RPE plays a major role in promoting angiogenesis in ocular vasculatures. Our finding that Rb1 inhibits the release of VEGF from RPE cells suggests that Rb1 has a significant role in the eye to protect against angiogenic diseases such as age-related macular degeneration.

Evidence for two retinoid cycles in the cone-dominated chicken eye.

In the classic retinoid cycle, 11-cis retinol is synthesized in the retinal pigment epithelium (RPE) by two enzymes: Isomerase I (RPE65) and lecithin:retinol acyltransferase (LRAT). The purpose of this study is to provide experimental evidence for two active isomerases in the cone-dominated chicken eye: an LRAT-dependent Isomerase I in the RPE and an ARAT (acyl CoA:retinol acyltransferase)-dependent isomerase (Isomerase II) in the retina. First, we show that whole chicken retina in vitro, removed from the RPE/choroid and sclera, produces 11-cis retinoids upon light exposure, indicating the existence of RPE-independent isomerase (Isomerase II) activity in the retina. Reverse transcriptase polymerase chain reaction studies show high levels of RPE65 expression in the RPE, low levels in the retina, and none in primary Muller cell cultures, indicating the presence of Isomerase I in the RPE and a minimal amount in the retina. Activities of the RPE and retina isomerases were then measured by enzyme assays with specific enzyme inhibitors. 2,2'-Bipyridine, a known Isomerase I inhibitor, and N-ethylmaleimide (NEM), a known LRAT inhibitor, significantly reduced Isomerase I activity but not Isomerase II activity. Progesterone, a known ARAT inhibitor, completely blocked Isomerase II activity but not Isomerase I activity. Thus, this study reports novel results for distinguishing the biochemical properties of Isomerase I from those of Isomerase II, as well a difference in their locations in the chicken eye. On the basis of these differences, the cone-dominated chicken eye must contain two retinoid cycles: a classic visual cycle for retinoid exchange between the RPE and the retina supported by Isomerase I in the RPE and an additional visual cycle for retinoid processing in the retina supported by Isomerase II.

Optical absorption and scattering of bovine cornea, lens and retina in the visible region.

Optical properties of bovine ocular tissues were determined at laser wavelengths in the visible region. The inverse adding doubling (IAD), Kubelka-Munk (KM), and inverse Monte Carlo (IMC) methods were applied to the measured values of the total diffuse transmission, total diffuse reflection, and collimated transmission to determine the optical absorption and scattering coefficients of the bovine cornea, lens and retina at 457.9 nm, 488 nm, and 514.5 nm laser lines from an argon ion laser. The optical properties obtained from these three methods were compared, and their validity is discussed.

A novel cone visual cycle in the cone-dominated retina.

The visual processing of humans is primarily reliant upon the sensitivity of cone photoreceptors to light during daylight conditions. This underscores the importance of understanding how cone photoreceptors maintain the ability to detect light. The vertebrate retina consists of a combination of both rod and cone photoreceptors. Subsequent to light exposure, both rod and cone photoreceptors are dependent upon the recycling of vitamin A to regenerate photopigments, the proteins responsible for detecting light. Metabolic processing of vitamin A in support of rod photopigment renewal, the so-called "rod visual cycle", is well established. However, the metabolic processing of vitamin A in support of cone photopigment renewal remains a challenge for characterization in the recently discovered "cone visual cycle". In this review we summarize the research that has defined the rod visual cycle and our current concept of the novel cone visual cycle. Here, we highlight the research that supports the existence of a functional cone-specific visual cycle: the identification of novel enzymatic activities that contribute to retinoid recycling, the observation of vitamin A recycling in cone-dominated retinas, and the localization of some of these activities to the Müller cell. In the opinions of the authors, additional research on the possible interactions between these two visual cycles in the duplex retina is needed to understand visual detection in the human retina.

Optical properties of ocular tissues in the near infrared region.

Near infrared characterization of optical properties of aqueous humor and vitreous humor of healthy human and bovine eyes has been performed. The indices of refraction (n) of these ocular tissues were determined using a Michelson interferometer. The total diffuse reflection (R (d)) and total transmission (T (t)) measurements had been taken for individual ocular tissue by using a double-integrating sphere setup and infrared laser diodes. The inverse adding doubling (IAD) computational method based on the diffusion approximation and radiative transport theory was applied to the measured values of n, R (d), and T (t) to calculate the optical absorption and scattering coefficients of the human and bovine ocular tissues. The scattering anisotropy value was determined by iteratively running the IAD method program and a Monte Carlo simulation of light-tissue interaction until the minimum difference in experimental and computed value for T (t) was realized. A comparison between the optical characterization of human and bovine ocular samples was also made.

11-cis-Acyl-CoA:retinol O-acyltransferase activity in the primary culture of chicken Muller cells.

A novel retinoid cycle has recently been identified in the cone-dominated chicken retina, and this cone cycle accumulates 11-cis-retinyl esters upon light adaptation. The purpose of this study is to investigate how 11-cis-retinyl esters are formed in the retina. Primary cultures of chicken Muller cells and cell membrane were incubated with all-trans- or 11-cis-retinol to study retinyl ester synthesis. In Muller cells, esterification of 11-cis-retinol was four times greater than esterification of all-trans-retinol. In the presence of palmitoyl-CoA and CRALBP, Muller cell membranes synthesized 11-cis-retinyl ester from 11-cis-retinol at a rate which was 20-fold higher than that of all-trans-retinyl ester. In the absence of CRALBP, 11-cis-retinyl ester synthesis was greatly reduced (by 7-fold). In the absence of palmitoyl-CoA, retinyl ester synthesis was not observed. Muller cell membranes incubated with radiolabeled palmitoyl-CoA resulted in the transfer of the labeled acyl group to retinol. This acyl transfer was greatly reduced in the presence of progesterone, a known ARAT inhibitor. 11-cis-ARAT activity remained unchanged when assayed in the presence of all-trans-retinol, suggesting a distinct catalytic activity from that of all-trans-ARAT. Apparent kinetic rates for 11-cis-ARAT were 0.135 nmol min(-)(1) mg(-)(1) (V(max)) and 11.25 microM (K(M)) and for all-trans-ARAT were 0.0065 nmol min(-)(1) mg(-)(1) (V(max)) and 28.88 microM (K(M)). Our data indicate that Muller cells in the chicken retina possess 11-cis-ARAT activity, thus providing an explanation for the accumulation of 11-cis-retinyl esters in the cone cycle.

Effect of light exposure on the accumulation and depletion of retinyl ester in the chicken retina.

A previous study has shown that in the cone-rich chicken retina, 11-cis retinyl ester increases with light exposure and decreases in subsequent dark adaptation. The purpose of this research is to study how light exposure (intensity and duration) determine the rate of accumulation and utilization as well as the size of this 11-cis retinyl ester pool in the chicken retina. Chickens were dark-adapted overnight before exposure to different light intensities and durations. Animals were sampled at regular time intervals. 11-cis retinal and 11-cis retinyl ester were extracted from the retina and analyzed by HPLC. An increase in light intensity from 1,000 and 2,000 Lux (for a 20 min exposure) increased the amount of 11-cis retinyl ester from 0.38 to 0.75 nmol/mg. An increase in the duration of light exposure from 10 to 20 min (at 2,000 Lux) also increased the amount of 11-cis retinyl ester in the chicken retina (from 0.37 to 0.75 nmol/mg). This 11-cis retinyl ester pool in the chicken retina was rapidly reduced to baseline level ( approximately 0.20 nmol/mg) upon dark adaptation. The rate of accumulation of 11-cis retinyl ester was dependent on light intensity and duration of exposure and the maximum rate was approximately 0.03 nmol/mg/min. In comparison, dark adaptation was associated with a significantly higher rate of 11-cis retinyl ester depletion (approximately 0.05), indicating that light exposure and dark adaptation were associated with different biochemical steps of retinoid storage and utilization. Results from this study are the first to show that the size of the 11-cis retinyl ester pool, as well as the rate of its accumulation and depletion in the cone-rich chicken retina, are determined by the intensity and duration of light exposure. These data support the suggestion that a light-driven cone cycle exists in the chicken retina.

Optical scattering, absorption, and polarization of healthy and neovascularized human retinal tissues.

The optical scattering, absorption, and polarization properties of human retinal tissues are investigated for a number of laser wavelengths in the visible range. The indices of refraction of these tissues are determined by applying Brewster's law. The inverse adding doubling method based on the diffusion approximation and radiative transport theory is applied to the measured values of total diffuse transmission, total diffuse reflection, and index of refraction to determine the optical absorption, scattering, and scattering anisotropy coefficients of the intact retinal tissues from healthy and diseased (neovascularized) human eyes. The polarization studies show that the retinal tissues possess significant intrinsic polarization characteristics, that are more pronounced in diseased tissues than in healthy tissues.

Retinoid cycles in the cone-dominated chicken retina.

In past decades, the role of retinoids in support of rod photopigment regeneration has been extensively characterized. In the rhodopsin cycle, retinal chromophore from bleached rod pigments is reduced to retinol and transferred to the retinal pigment epithelium (RPE) to store as all-trans retinyl ester. This ester pool is subsequently utilized for visual pigment regeneration. However, there is a lack of information on the putative cone visual cycle. In the present study, we provide experimental evidence in support of a novel retinoid cycle for cone photopigment regeneration. In the cone-rich chicken, light exposure resulted in the accumulation of 11-cis retinyl esters to the retina and all-trans retinyl esters to the RPE. Both the rate of increase and the amount of 11-cis retinyl esters in the retina far exceeded those of the all-trans retinyl esters in the RPE. In response to dark adaptation, this 11-cis retinyl ester pool in the retina depletes at a rate several times faster than the all-trans retinyl ester pool in the RPE. In vitro, isolated, dark-adapted retinas devoid of RPE show both an accumulation of 11-cis retinyl ester and a concomitant reduction of 11-cis retinal chromophore in response to light exposure. Finally, we provide experimental results to elucidate a cone visual cycle in chicken by relating the change in retinoids (retinal and retinyl ester) with time during light and dark adaptation. Our results support a new paradigm for cone photopigment regeneration in which the 11-cis retinyl ester pool in the retina serves as the primary source of visual chromophore for cone pigment regeneration.

Optical characterization of bovine retinal tissues.

An in-depth characterization of the optical properties of bovine retinal and retinal pigment epithelium-choroidal tissues has been performed. The indices of refraction of these ocular tissues were determined by applying Brewster's law. The inverse adding doubling method based on the diffusion approximation and radiative transport theory is applied to the measured values of the total diffuse transmission, total diffuse reflection, and collimated transmission to calculate the optical absorption, scattering, and scattering anisotropy coefficients of the bovine retinal and retinal pigment epithelium-choroidal tissues. The values of the optical properties obtained from the inverse adding doubling method are compared with those generated by the Monte Carlo simulation technique. Optical polarization measurements are also performed on bovine retinal tissues. Our studies show that both retina and retinal pigment epithelium-choroid possess strong polarization characteristics.

The hepatic retinyl ester hydrolase activity is depressed at the onset of diabetes in BB rats.

Dietary vitamin A as retinyl ester is hydrolysed and re-esterified with long-chain fatty acids in the small intestine. The esterified vitamin A is subsequently stored in the liver, where it is hydrolysed to free retinol to be transported by carrier proteins to the target tissue. A decreased availability of retinol carrier proteins has been suggested to be responsible for affecting metabolic availability of vitamin A in type 1 diabetes. Using BB Wistar rats, the present study was undertaken to examine whether the presence of a hyperglycaemic state modifies retinyl ester hydrolase (REH) activity in the intestine and the liver. At the onset of diabetes, hepatic REH enzymatic activity was significantly decreased. However, REH activity remained unaffected in the small intestine, including both ileum and jejunum. Diabetes also resulted in decreased plasma and liver concentrations of retinol. An in vitro study was conducted to examine the effect of diabetes on the intestinal uptake of retinyl palmitate. Jejunum and ileum from diabetic and non-diabetic BB rats were incubated with labelled retinyl palmitate at different concentrations ranging from 32 to 256 nmol/l. The uptake of retinyl palmitate was increased in both diabetic and non-diabetic rats together with the increase of substrate concentration. However, no significant difference was observed in the uptake of retinyl palmitate between diabetic and non-diabetic rats. These present results suggest that the depressed hepatic REH activities may contribute to the diabetes-associated metabolic derangement of vitamin A.

Vitamin A Homeostasis in the Diabetic Rat.

The concentrations of vitamin A (retinol) and retinyl ester in the plasma and liver of normal and diabetic rats were measured by HPLC (high-performance liquid chromatography). Diabetic rats had severe hyperglycemia, induced by a single streptozotocin injection 5 weeks prior to sampling. In the normal rats, plasma retinyl palmitate was very low, and the level was increased 10-fold by diabetes. Detailed time-course studies showed that rats became hyperglycemic within 48 h of streptozotocin injection, yet the plasma retinyl palmitate level was not elevated until some three weeks later. Severe diabetes did not significantly influence plasma retinol: however. free retinol in the liver was elevated within 10 days of initiation of the disease and continued to increase for the duration of the study. These results show that streptozotocin-induced diabetes significantly alters the concentrations of hepatic retinol and plasma retinyl ester. The biochemical mechanism(s) of this altered vitamin A homeostasis in diabetes and its possible relationship to tissue pathogenesis are not known at present.

RETINOID METABOLISM AND CONVERSION OF RETINOL TO DEHYDRORETINOL IN THE CRAYFISH (PROCAMBARUS CLARKII) RETINA.

Activities of retinylester hydrolase (REHase) and retinol dehydrogenase (RDHase) were detected in the crayfish retina which has two visual pigment chromophores and three stored retinoids.RDHase and 11-cis REHase activities were localized to rhabdomal fraction and enzymes were specific to 11-cis isomers of substrates. Overall reaction from retinylester to retinal was highly specific to 11-cis isomers and this reaction was not selective between retinol and dehydroretinol.When 11-cis [3H]-retinol was incubated with retina homogenates, radioactivity was detected in 11-cis dehydroretinol.These results suggest that retinol is converted to dehydroretinol after hydrolysis of retinylesters.

An evaluation on the level of retinoids in the bovine pineal body.

Using high performance liquid chromatography, the level of retinoids was determined in the bovine pineal gland, retina, retinal pigment epithelium, cortical and subcortical brain tissues, skeletal muscle and the liver. Similar to the retina, the bovine pineal gland possesses levels of retinol and retinyl esters significantly higher than other brain tissues and muscle. These results are in agreement with the suggestion that the mammalian pineal gland and the retina may be of similar phylogenetic origin. However, unlike the retina, the bovine pineal gland does not possess any detectable level of retinal, the chromophore for visual pigments in the retina. This finding suggests that the bovine pineal gland does not possess rhodopsin nor the property of phototransduction which has been fully established in the retina and pineal glands of lower vertebrates.

Distribution of retinoids and 3,4-didehydroretinoids in the goldfish.

Using high-performance liquid chromatography, the quantity of retinol, retinal, and retinyl esters as well as their 3,4-didehydro derivatives were measured in several body compartments and in the remainder of the body of 11 goldfish. Eighty-six percent of these retinoids and 3,4-didehydroretinoids existed as retinyl and 3,4-didehydroretinyl esters, 10% as retinal and 3,4-didehydroretinal, and 4% as retinol and 3,4-didehydroretinol. Most (94%) of these retinoids and 3,4-didehydroretinoids were found in ocular tissues and high ratios of 3,4-didehydroretinoids/retinoids were observed in most tissue extracts. These data suggest that in the goldfish ocular tissues are important locations of retinoid and 3,4-didehydroretinoid metabolism, which favors the formation and (or) storage of 3,4-didehydroretinoids.