Diabetic retinopathy in pregnancy.
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Diabetic Retinopathy↗
Biomedical subjects
U Chakravarthy
Publications and source records attributed to U Chakravarthy.
Teletherapy in the management of patients with age-related macular degeneration complicated by subfoveal neovascularisation: an overview.
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Dose-Response Relationship, Radiation↗
Endothelin expression in ocular tissues of diabetic and insulin-treated rats.
PURPOSE: The endothelins are potent vasoactive peptides that are widely distributed in ocular tissues. There is evidence linking the endothelins to vascular dysfunction in diabetic microangiopathy. Thus, the synthesis and distribution of endothelin-1 (ET-1) and endothelin-3 (ET-3) were studied in the retinas of diabetic and nondiabetic animals. METHODS: Levels of ET-1 and ET-3 were determined by radioimmunoassay in ocular tissues of normal rats, and in rats with streptozotocin-induced diabetes of 6 and 12 weeks' duration, insulin-treated and untreated. In a separate cohort of similarly treated animals, retinal vascular trypsin digest preparations were immunostained, using antibodies raised against ET-1 and ET-3. RESULTS: Ocular ET-1 levels were elevated twofold in diabetic animals that received insulin treatment for 7 days when compared with levels in normal rats. Insulin treatment for 10 days before death caused a fourfold elevation of ET-1 in ocular tissues. Endothelin-1 was also increased in 12-week-old diabetic animals and in those maintained on insulin throughout their period of diabetes. Immunofluorescence to anti-ET-1 within the capillary bed and veins of the retina in diabetic insulin-treated animals was elevated when compared with digests from normal litter-matched control animals. Ocular tissue ET-3 levels were unaffected by diabetes. CONCLUSIONS: Overall ocular and retinal tissue levels of ET-1 were selectively elevated by diabetes and insulin treatment, suggesting that the endothelins may be involved in the pathogenesis of diabetic retinal microangiopathy.
Animals↗
Nitric oxide synthase and nitrite production in human spermatozoa: evidence that endogenous nitric oxide is beneficial to sperm motility.
The aim of this study was to investigate the presence of nitric oxide synthase (NOS) and the production of nitric oxide (NO) by human spermatozoa. Immunoreactivity was examined using a polyclonal antibody raised against porcine cerebellar nitric oxide synthase and monoclonal endothelial (eNOS) and brain (bNOS) antibodies. Using each antibody, NOS was observed localized in the head and midpiece regions of the spermatozoon. Immunofluorescence observed for eNOS and bNOS was more intense in normozoospermic samples. Sperm motility was assessed by computer-assisted semen analysis (CASA) in the presence and absence of NG-nitro-L-arginine methyl ester (L-NAME; 10(-5)M), and NO synthesis inhibitor or tumour necrosis factor (TNF)-alpha (20 IU/ml), a superoxide inducer. In the presence of L-NAME, percentage progressive motility, average path velocity (VAP), straight line velocity (VSL) and curvilinear velocity (VCL) were significantly reduced after 30 min. Sperm viability was not decreased by TNF alpha or L-NAME. The accumulation of nitrite (the stable end-product of the NOS/NO pathway) by spermatozoa was measured using the Griess reaction. After 8 h, nitrite concentrations were lower in asthenozoospermic compared to normozoospermic samples. In the presence of TNF alpha, nitrite accumulation was significantly reduced in normozoospermic samples. We conclude that NOS is present in human spermatozoa and that eNOS and bNOS are abundant in normozoospermic samples. Nitric oxide (at endogenous concentrations) appears to be necessary for adequate sperm motility.
Animals↗
Teletherapy for subfoveal choroidal neovascularisation of age-related macular degeneration: results of follow up in a non-randomised study.
AIM: A preliminary report indicated stable or improved vision in 12 of 19 patients with subfoveal choroidal neovascularisation treated with 12 or 15 Gy of 6 MV photons to the affected macula after an average follow up of 18 months. Here the prolonged follow up findings in this group of treated patients is reported which was further increased to 41. METHODS: Forty one patients with subfoveal choroidal neovascularisation were treated with 10, 12, or 15 Gy of 6 MV photons to the macula of the affected eye. Thirteen eyes of 12 patients were also observed as a non-randomised comparison group. RESULTS: At 12, 18, and 24 months of follow up the mean change in visual acuity in eyes treated with radiotherapy was less than 1 Bailey-Lovie line from that measured at presentation. By contrast, the eyes in the comparison group lost 3.7 lines of acuity at 12 months which increased to 4.5 at 24 months. These differences were highly significant at each of the time points. When initial visual acuity was taken into account, treated eyes lost on average 12% of baseline acuity throughout follow up, whereas eyes belonging to the untreated group lost 50% of baseline acuity at 1 year, and 75% at 2 years. There was no significant difference in visual outcome between the three dose regimens used, which may simply be a reflection of the small sample size in each group. There was no evidence of radiation induced retinopathy or optic neuropathy in any treated patients. CONCLUSIONS: Teletherapy appeared to have a treatment effect in eyes with subfoveal choroidal neovascularisation resulting in maintained visual function without significant radiation induced morbidity.
Aged↗
Endothelin-like immunoreactivity and receptor binding in the choroid and retina.
This study has examined the localisation and receptor-binding of the endothelins in retina and choroid of human and rat origin. Immunoreactivity to anti-ET1 and anti-ET3 was investigated in trypsin digests, frozen sections and ultrathin sections using immunocytochemistry and immunogold labelling techniques. In addition, receptor binding of 125I-ET1 and 125I-ET3 was visualised and quantified using autoradiography and image analysis. Intense immunoreactivity to anti-ET1 and anti-ET3 was observed in the photoreceptor inner segments and in the outer plexiform layer (OPL) of human and rat retina. Ultrastructural localisation using immunogold labelling confirmed the presence of ET1 and ET3 in the photoreceptor cells. In retinal vascular digests, ET1 was visualised in the arteries, arterioles and at the pre-arteriolar sphincters, however, immunoreactivity to anti-ET3 was absent in the retinal vasculature. Both ETA and ETB-type receptor binding sites to 125I-ET1 and 125I-ET3 were detected in the vascular smooth muscle of choroidal and retinal vessels with the former being predominant. Extravascular binding sites of the ETB-type were found in the ganglion cell layer.
Animals↗
Receptor binding and biologic activity of synthetic ET-1 peptides in the retinal pericyte.
PURPOSE: The purpose of this study was to examine the effect of synthetic endothelin (ET)-1 peptides with antigenic potential for binding and biologic activity using an in vitro model of microvascular pericytes. METHODS: All possible sequential hexapeptide fragments of endothelins -1, -2, and -3 were synthesized on polyethylene rods and tested for reactivity with antibodies for ET-1 and ET-3. The most highly antigenic peptide ET-1[3-8] and the least antigenic ET-1[1-6], which gave low reactivity in the enzyme-linked immunosorbent assay (ELISA), were synthesized. The C-terminal hexapeptide ET-1[16-21], which has been reported as an ETB receptor agonist but which gave no reactivity in the ELISA, also was investigated. The synthesized ET analogues were tested for receptor binding, inositol phosphate generation, and mitogenesis in bovine retinal pericytes. RESULTS: ET-1[16-21] partially inhibited both [125I]-ET-3 binding at high concentrations, as did ET-1[1-6]. In contrast, ET-1[3-8] displaced labeled ET-1 binding but not labeled ET-3 binding. None of the peptides had any significant mitogenic or second-messenger responses when compared to those elicited by the full ET-1 molecule. CONCLUSIONS: The result of the current work shows that the sequence, ET[3-8], is involved in isopeptide-specific binding to ETA receptors, but it suggests that other regions of the molecule are necessary for full bioactivity in microvascular pericytes.
Animals↗
Increased endocytosis in retinal vascular endothelial cells grown in high glucose medium is modulated by inhibitors of nonenzymatic glycosylation.
We sought to determine if hyperglycaemia is responsible for increased retinal vascular endothelial-cell (RVEC) endocytosis in diabetes and to assess the role of nonenzymatic glycosylation in mediation of this novel endothelial-cell pathology. RVECs were propagated in media containing either 5 or 25 mmol/l glucose for up to 10 days after which they were exposed to the protein tracer horseradish peroxidase for 30 min. The level of RVEC endocytosis was quantified in intact cell monolayers by electron microscopic stereology, and in cell lysates by a simple spectrophotometric method. The effect of the nonenzymatic glycosylation inhibitors, aminoguanidine and D-lysine, on high-glucose medium induced changes in RVEC endocytosis was tested by inclusion of these agents in the culture medium. RVECs exposed to 25 mmol/l glucose showed a stepwise increase in endocytosis of horseradish peroxidase culminating in a two- to threefold increase after 10 days. Endocytosis returned to normal levels after a further 10 days in 5 mmol/l glucose medium. The increase in RVEC endocytosis was markedly reduced, but not completely normalised, by aminoguanidine and D-lysine. Exposure of cultured RVECs to 25 mmol/l glucose causes an increase in endocytosis of similar magnitude to that experienced by RVEC in early diabetes, and implicates hyperglycaemia in the latter situation. A significant component of the increase in RVEC endocytosis appears to be mediated by nonenzymatic glycosylation.
Animals↗
Asymmetry of disciform scarring in bilateral disease when one eye is treated with radiotherapy.
AIMS/BACKGROUND: A previous study has shown that in age-related macular degeneration a high degree of concordance of disciform scar size occurs in the two eyes of any one patient. In a study of 35 patients with choroidal neovascular membrane who were treated with low dose ionising radiation to the macula of the affected eye, 11 were found to have bilateral disease. METHODS: The visual outcome and scar size and morphology in the two eyes of each of these patients were compared. RESULTS: In all radiotherapy treated eyes the appearance of the scar ranged from subtle subretinal pigmentary changes to dense subretinal fibrosis. By contrast all untreated fellow eyes showed marked subretinal scarring. Scars in radiotherapy treated eyes occupied an area that was approximately one third of that in untreated fellow eyes (3.8 mm2 v 11.7 mm2). Distance and near visual acuities in radiotherapy treated eyes were significantly better than that of untreated fellow eyes (p < 0.0033). Although untreated fellow eyes of necessity had longer follow up periods (64.5 months), the mean follow up time in treated eyes was 28 months by which time the disciform response is generally thought to have ceased evolving. CONCLUSION: This study has provided evidence in support of reduced scarring and maintenance of better central visual function in radiotherapy treated eyes when compared with untreated fellow eyes.
Aged↗
Nitric oxide synthase activity and expression in retinal capillary endothelial cells and pericytes.
The purpose of this study was to examine nitric oxide synthase (NOS) expression in the retinal vasculature in vivo and to study nitric oxide (NO) synthesis in vitro in retinal microvascular endothelial cells and pericytes. Immunoreactivity was examined using a polyclonal antibody raised against porcine cerebellar nitric oxide synthase on frozen sections cut from postmortem human retina and trypsin digests of rat retinal vasculature. The synthesis of nitrite, a stable end product from the interaction of NO with molecular oxygen, was measured in culture supernatants of retinal microvascular cells under basal and stimulated conditions. Expression of constitutive NOS (cNOS) in these cells was examined using the polymerase chain reaction (PCR). Strong NOS immunoreactivity was seen in the endothelium of choroidal and retinal vessels. Nitrite synthesis was documented in supernatants from cultured microvascular endothelial cells which increased significantly following exposure to A23187 and cytokines. Nitrite synthesis by pericytes was not detectable under basal conditions or following stimulation with A23187. Bacterial lipopolysaccharide (LPS), a potent inducer of NOS, caused an increase in nitrite concentrations in pericyte supernatants 24 h after stimulation suggesting the presence of inducible NOS (iNOS). PCR amplification confirmed the presence of the cNOS gene in endothelial cells but not in pericytes. Retinal vascular endothelial cells express significant amounts of NOS constitutively in vivo and in vitro which is activated by Ca++. Also, endothelial cells can be stimulated to synthesize iNOS by cytokines. Retinal pericytes too show iNOS activity following exposure to bacterial LPS. These results suggest that the nitric oxide synthase/nitric oxide pathway may be involved in the regulation of microcirculatory haemodynamics in the retina.
Amino Acid Oxidoreductases↗
Characterization of endothelin A (ETA) and endothelin B (ETB) receptors in cultured bovine retinal pericytes.
PURPOSE: The endothelins are a family of structurally similar vasoactive peptides. It has been shown recently that cultured retinal microvascular endothelial cells secrete endothelin-1 (ET-1) and that corresponding pericytes bear receptors and are responsive to this peptide. These findings suggest a role for ET-1 in the autoregulation of retinal blood flow. There are at least two known subtypes of ET receptors, ETA and ETB. The purpose of this study was to characterize endothelin receptor subtypes on cultured bovine retinal pericytes (BRP). METHODS: To characterize the specific binding sites for ET-1 and ET-3 on monolayers of BRP, a radioligand binding assay was performed using [125I] ET-1 and [125I] ET-3. Competition binding studies with ET-1 and ET-3 were used to assess the heterogeneity of the ET-receptor population on BRP. Also, [125I] ET-1 and ET-3 were covalently linked to their corresponding receptors and analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography. RESULTS: [125I] ET-1 and [125I] ET-3 showed specific binding to BRP and subsequent Scatchard analysis for both labels showed upward concavity, implying two-site ligand binding. Unlabeled ET-1 was found to displace [125I] ET-1 with greater efficiency than ET-3, indicating the presence of the ETA receptor subtype. Conversely, [125I] ET-3 was displaced by ET-1 and ET-3 with equal potency, indicating a component of ETB in the receptor population. Preincubation with BQ123, an ETA selective antagonist, decreased the binding of [125I] ET-1 but had no effect on [125I] ET-3 binding curves. Affinity cross-linking of the receptors showed two distinct protein bands on SDS-PAGE of 66 and 45 kd, corresponding to ETA and ETB. CONCLUSIONS: These results show that BRP possess ETA and ETB receptor subtypes. The function of ETB on BRP may be to modulate the vasoconstrictive effect of ET-1 caused through ETA.
Animals↗
Molecular characterization of endothelin receptors and the effect of insulin on their expression in retinal microvascular pericytes.
Microvascular pericytes contain predominantly endothelin A (ET(A))-like binding sites and also a smaller number of ETB binding sites. In this study we verified the expression of both receptors in these cells. We then examined the effect of insulin, a potent mediator of vasodilatation, on the expression of these receptors in pericytes by Northern hybridization. Northern hybridization studies showed that ET(A) mRNA levels were not influenced by exposure to insulin (1 x 10(-7) M). By contrast, ETB receptor mRNA, which was minimal under basal conditions, was significantly increased by 4 h after exposure to insulin.
Animals↗
Quantitative analysis of hyaluronan in vitreous humor using capillary electrophoresis.
Capillary electrophoresis was used for the separation and quantitative analysis of the glycosaminoglycan hyaluronan in human and bovine vitreous with detection by UV absorbance at 200 nm. Calibration was carried out using standards made up from known concentrations of hyaluronan of umbilical cord origin. The purity of the standard was examined by 1H NMR (nuclear magnetic resonance). Concentrations as low as 25 micrograms/mL could be detected by capillary electrophoresis. Confirmation that the signal was due to hyaluronan was obtained by depolymerization of the native mucopolysaccharide by hyaluronidase. This resulted in loss of the hyaluronan peak and appearance of several new peaks corresponding to the oligomeric fragments which had shorter migration times. Capillary electrophoresis is a reproducible and sensitive technique for the quantification and characterisation of hyaluronan in vitreous samples.
Animals↗
Altered endothelin-1 induced contraction and second messenger generation in bovine retinal microvascular pericytes cultured in high glucose medium.
The effect of simulated hyperglycaemia on bovine retinal pericytes was studied following culture of these cells for 10 days under normal (5 mmol/l) and elevated (25 mmol/l) glucose conditions in the absence of endothelial cells. Pericytes cultured under high ambient glucose exhibited both a delayed and reduced contractile response following stimulation with endothelin-1. Stimulation with 10(-7) mol/l endothelin-1 for 30 s caused significant contraction in cells grown in both 5 mmol/l and 25 mmol/l glucose. The former also contracted significantly with 10(-8) mol/l endothelin-1. Further, at all concentrations tested, statistical comparison of the time course of contraction showed a significant difference (p < 0.02) in the reduction of planimetric surface area between the two cell groups. Since neither binding of endothelin-1 nor the number of receptors for this peptide were significantly different (p > 0.1) between bovine retinal pericytes grown for 10 days under normo- or hyperglycaemic conditions, it became apparent that the altered contractility in bovine retinal pericytes following culture in high glucose must be due to post-binding intracellular disturbance(s). Indeed, both basal and 15 s post-stimulation with 10(-8) mol/l endothelin-1, levels of inositol trisphosphate were significantly reduced (p < 0.05 and p < 0.02, respectively) in pericytes cultured for 10 days in 25 mmol/l glucose. These results show that endothelial-independent alterations in contractility of pericytes occur when they are grown in conditions which simulate hyperglycaemia. The results also suggest that the observed attenuation in response to endothelin-1 stimulation evident in pericytes grown under simulated hyperglycaemic conditions is not due to alterations in peptide binding.
Animals↗
Immunoreactive endothelin distribution in ocular tissues.
PURPOSE: To study the distribution of the endothelins in the eye using ocular tissues of human, rat, and porcine origin. METHODS: Extracts of ocular tissues were examined for immunoreactivity to endothelin 1 and 3 and pro-endothelin 1 using radioimmunoassay. Characterization of immunoreactivity was verified using high-performance liquid chromatography. RESULTS: Immunoreactivity to endothelin 1 and endothelin 3 was found in all ocular tissues except the cornea, which contained no immunoreactivity to endothelin 3. Highest levels were found in the choroid, although species-related differences were present. Immunoreactivity to endothelin 3 was generally twofold to threefold higher than immunoreactivity to endothelin 1. The majority of endothelin-like-immunoreactivity in the retina was blood vessel-associated. High-pressure liquid chromatography (HPLC) confirmed that immunoreactive endothelin 1 and endothelin 3 in the tissue extracts eluted in identical positions to their respective standard synthetic peptides. CONCLUSIONS: The endothelins are abundantly distributed in the eye. Endothelin 1 is present in its mature, 21 amino acid form, and only minimal amounts of the precursor pro-endothelin is found in ocular tissues. The wide distribution patterns point to complex roles for these peptides in blood vessel physiology and in such other functions as regulation of aqueous outflow, and in neurotransmission or modulation.
Aged↗
Tachykinin-1 gene products in porcine ocular tissues: evidence for transcriptional and post-translational regulation.
The tachykinin-1 gene in mammals produces structurally-related regulatory peptides, substance P (SP), neurokinin A (NKA), neuropeptide K (NPK) and neuropeptide-gamma. The production of these peptides is regulated by both differential mRNA transcription and post-translational precursor processing. Such processes are known to be highly tissue- and species-specific. In this study, we have examined tachykinin-1 gene expression and precursor processing in porcine ocular tissues by employing specific tachykinin radioimmunoassays coupled with reverse phase HPLC characterization. Optic nerve, cornea, iris, ciliary body, retina, choroid and sclera were micro-dissected from freshly enucleated porcine eyes (n = 10). Following acidified ethanol extraction of tissues, dried extracts were reconstituted and subjected to two radioimmunoassays, one of which is highly specific for intact SP, the other for NKA, NKB, NPK and neuropeptide-gamma. In all tissue extracts except the retina, the molar concentration of SP immunoreactivity was significantly greater than that of NKA. These data would imply expression of both alpha- and beta-preprotachykinin-1 in these ocular tissues. Reverse phase HPLC analysis confirmed the presence of authentic SP and NKA in all tissue extracts. However, in extracts of the retina, NKA immunoreactivity co-eluted with synthetic NPK standard. These chromatographic data suggest differential processing of the beta-preprotachykinin-1 precursor in the retina compared with the other ocular tissues. Thus differential mRNA transcription of the tachykinin-1 gene coupled with differential precursor processing appears to occur in porcine ocular tissues and may be a process of functional significance in the regulation of visual physiology.
Animals↗
Treatment of age-related subfoveal neovascular membranes by teletherapy: a pilot study.
This investigation was designed to determine whether low dose radiation to the macular region could influence the natural course of age-related subfoveal neovascularisation. Nineteen patients with subfoveal membranes due to age-related macular degeneration (ARMD) were treated with 10 or 15 Gy of 6 MV photons and seven patients who declined treatment were followed up as controls. Six controls and all treated patients had completed follow up times of at least 12 months. Visual acuity was maintained or improved in 78% and 63% of treated patients at their 6 and 12 month follow up examinations respectively. By contrast visual acuity showed steady deterioration in six of seven controls. Significant neovascular membrane regression, as measured by image analysis, was recorded in 68% and 77% of treated patients at 6 and 12 months post-radiation, whereas the membranes in all seven control patients showed progressive enlargement. This study suggests that low doses of radiation can maintain central vision and induce regression of subfoveal neovascular membranes of ARMD in a significant proportion of patients. We now believe it appropriate to proceed to a prospective randomised study to test this hypothesis further.
Age Factors↗
The effect of endothelin 1 on the retinal microvascular pericyte.
The effect of the highly vasoactive peptide endothelin 1 (ET1) was tested on bovine retinal microvascular pericytes propagated in vitro. Specific binding of 125I-ET1 to retinal pericytes was documented by autoradiography. ET1 caused contraction of pericytes at a concentration of 0.1 nM which was accompanied by increases in inositol phosphates. Exposure of pericytes to 10 nM ET1 resulted in the aggregation and realignment of muscle-specific actins into bundles which were oriented parallel to the long axis of the cell, and ET1 was also mitogenic to pericytes in the presence of low levels of fetal calf serum. These observations suggest that ET1 may play an important role in endothelial cell-pericyte interactions within the microvasculature of the retina and that it may be involved in the autoregulation of retinal blood flow.
Actins↗