endothelin-1 and Diabetic-Retinopathy

Increasing evidence suggests that the complex interactions among multiple cell types including neuronal, glial, and vascular cells, are critical for maintaining adequate cerebral blood flow that is necessary for normal brain function and survival. The disturbance of these interactions contributes to the pathogenesis of central nervous system disorders such as stroke and Alzheimer's disease. The retina is part of the central nervous system, and the properties of vasculature in the retina are similar to those in the brain. The interactions among multiple cell types in the retina also play an important role in the maintenance of tissue homeostasis, and the impairment of interactions can contribute to the onset and/or progression of retinal diseases. In this review, we describe the neurovascular interactions in the retina and alternations of interactions in pathological conditions such as diabetic retinopathy and glaucoma.

Topics: Angiotensin II; Catecholamines; Cell Communication; Diabetic Retinopathy; Disease Progression; Drug Discovery; Endothelin Receptor Antagonists; Endothelin-1; Glaucoma; Homeostasis; Molecular Targeted Therapy; N-Methylaspartate; Neuroglia; Neurons; Nitric Oxide; Nitric Oxide Synthase Type II; Potassium Channels, Calcium-Activated; Receptors, GABA; Retina; Retinal Diseases; Retinal Vessels

Most of the late diabetic complications such as retinopathy, nephropathy, and neuropathy, have their basis in disturbed microvascular function. Structural and functional changes in the micro-circulation are present in diabetes mellitus irrespective of the organ studied, and the pathogenesis is complex. Endothelial dysfunction, characterized by an imbalance between endothelium-derived vasodilator and vasoconstrictor substances, plays an important role in the pathogenesis of diabetic microangiopathy. Increased circulating levels of endothelin-1 (ET-1), a potent vasoconstrictor peptide, has been found in patients with diabetes, and a positive correlation between plasma ET-1 levels and microangiopathy in patients with type 2 diabetes has been demonstrated. In addition to its direct vasoconstrictor effects, enhanced levels of ET-1 may contribute to endothelial dysfunction through inhibitory effects on nitric oxide (NO) production. Vascular endothelial dysfunction may precede insulin resistance, although the feature of insulin resistance syndrome includes factors that have negative effects on endothelial function. Furthermore, ET-1 induces a reduction in insulin sensitivity and may take part in the development of the metabolic syndrome. In the following, the mechanisms by which ET-1 contributes to the development of diabetic microangiopathy and the potentially beneficial effect of selective ET(A) receptor antagonists are discussed.

Topics: Cardiovascular Agents; Diabetic Angiopathies; Diabetic Nephropathies; Diabetic Neuropathies; Diabetic Retinopathy; Endothelin Receptor Antagonists; Endothelin-1; Humans; Microcirculation; Receptors, Endothelin; Treatment Outcome; Up-Regulation

Dysfunction and death of microvascular cells and imbalance between the production and the degradation of extracellular matrix (ECM) proteins are a characteristic feature of diabetic retinopathy (DR). Glucose-induced biochemical alterations in the vascular endothelial cells may activate a cascade of signaling pathways leading to increased production of ECM proteins and cellular dysfunction/death. Chronic diabetes leads to the activation of a number of signaling proteins including protein kinase C, protein kinase B, and mitogen-activated protein kinases. These signaling cascades are activated in response to hyperglycemia-induced oxidative stress, polyol pathway, and advanced glycation end product formation among others. The aberrant signaling pathways ultimately lead to activation of transcription factors such as nuclear factor-kappaB and activating protein-1. The activity of these transcription factors is also regulated by epigenetic mechanisms through transcriptional coactivator p300. These complex signaling pathways may be involved in glucose-induced alterations of endothelial cell phenotype leading to the production of increased ECM proteins and vasoactive effector molecules causing functional and structural changes in the microvasculature. Understanding of such mechanistic pathways will help to develop future adjuvant therapies for diabetic retinopathy.

Topics: Animals; Basement Membrane; Capillaries; Cell Death; Diabetic Retinopathy; Endothelin-1; Endothelium, Vascular; Enzyme Activation; Extracellular Matrix; Glucose; Humans; Hyperglycemia; Protein Kinase C; Signal Transduction; Vasoconstriction; Vasodilation

Endothelin-1 (ET-1) is a 21 amino acid peptide originally purified from conditioned medium of cultures of porcine aortic endothelial cells. It is now known that there are three endothelin genes in the human genome (ET-1, ET-2, and ET-3 genes). ET-1 and ET-2 are both strong vasoconstrictors, whereas ET-3 is a potentially weaker vasoconstrictor compared to the other two isoforms. Besides being a very potent vasoconstrictor, ET-1 also acts as a mitogen on the vascular smooth muscle and thus it may play a role in the development of vascular diseases. There is evidence that impaired auto-regulation of blood flow is involved in the pathogenesis of diabetic microangiopathy. It is known that the ability of the diabetic's circulation to distribute blood is affected, especially during increased blood flow. In most tissues this causes no serious burden, but three tissues are usually susceptible to disturbance. They are the retina, renal cortex, and peripheral nerves. Retinal vascular auto-regulation is defined as the ability of the blood vessels to keep blood flow constant under varying perfusion pressure in order to match it to tissue oxygen and metabolic requirements. The failure of auto-regulation is an important and often early feature of diabetic retinopathy. Since human retina vessels lack extrinsic innervation, retinal vessel calibre and local blood flow are normally regulated by non-nervous mechanisms intrinsic to the retina. There is now a considerable body of evidence suggesting that retinal pericytes are the main regulators of vascular tone in the retinal capillaries because they contain components of contractile proteins similar to vascular smooth muscle cells and because they also possess ET-1 receptors. Furthermore. ET-1 has been shown to cause vasoconstriction of retinal vessels as well as to have mitogenic effects on retinal pericytes. Hence, alterations in the pericyte-ET interaction may have a role causing early hemodynamic and histopathological abnormalities found in diabetic retinopathy. On the contrary, Chakrabarti et al. demonstrate that retinas from the chronic diabetic BB/W rats (6 months) show an increase in ET-1, ET-3, ET(A) receptor and ET(B) receptor mRNA expressions when compared to those from control rats. Similar results are noted by them using immunohistochemical methods. Finally, an increased ocular, and retina tissue levels of ET-1 in diabetic rats have also been reported by Chakravarthy et al., as well as by Takagi et al. All of th

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Diabetic Retinopathy; Endothelin-1; Endothelin-2; Endothelin-3; Humans; Retina; Retinal Vessels

To determine the benefits of calcium dobesilate (CaD) administration on endothelial function and inflammatory status in patients with diabetic retinopathy through measurement of serum levels of endothelin-1 and high-sensitivity C-reactive protein (hsCRP).. In a double-blind, randomized clinical trial, 90 patients with either severe nonproliferative or proliferative diabetic retinopathy and with blood glucose level of 120-200 mg/dl were randomly allocated to treatment with either CaD tablets (500 mg daily) or placebo for 3 months. Visual acuity, intraocular pressure, and macular status were performed before the study. The serum levels of endothelin-1 and hsCRP were evaluated in both groups before and at the third month of the trial.. The median serum level of hsCRP significantly differed between the groups 3 months following the CaD or placebo administration (2.2 mg/l in the CaD group versus 3.7 mg/l in the placebo group, p=0.01). The mean endothelin-1 serum level was 0.69±0.32 pg/ml in the CaD group and 0.86±0.30 pg/ml in the placebo group (p=0.01). Furthermore, in the CaD group, the serum levels of both endothelin-1 and hsCRP were significantly decreased 3 months after administration of CaD (p<0.001).. Administration of the CaD in the patients with diabetic retinopathy may reduce the serum levels of endothelin-1 and hsCRP. This might imply amelioration of the endothelial function and inflammatory status following CaD therapy in these patients.

Topics: Aged; C-Reactive Protein; Calcium Dobesilate; Diabetic Retinopathy; Double-Blind Method; Endothelin-1; Female; Hemostatics; Humans; Lipids; Male; Middle Aged; Retinal Vessels

The aim of the study was to evaluate whether normoalbuminuric type 1 diabetic patients with diabetic retinopathy (DR) have an impaired tubular response to desmopressin (dDAVP, a synthetic analog of vasopressin) administration, and its relationship with plasma and urine endothelin-1 (ET-1) levels.. This was an interventional case-control study.. The study was conducted at a referral center.. Fifteen normoalbuminuric type 1 diabetic patients with DR were compared with 30 normoalbuminuric type 1 diabetic patients without DR. Both groups were matched by age, gender, body mass index, glycosylated hemoglobin, and the main laboratory markers of kidney function.. After a 12-h period of water deprivation, dDAVP (0.3 microg/kg) was infused over 20 min. Urine was collected at baseline and 1, 2, and 3 h after dDAVP administration. ET-1 was assessed by ELISA.. dDAVP induced a lower rise in urine osmolality in patients with DR (from 650 +/- 206 to 754 +/- 224 mosmol/kg; P = 0.02) than in diabetic patients without DR (from 714 +/- 194 to 905 +/- 163 mosmol/kg; P < 0.0001). In addition, fractional excretion of Na+ decreased in patients without DR (from 0.45 +/- 0.30 to 0.29 +/- 0.29%; P = 0.04) but not in the diabetic patients with DR (from 0.36 +/- 0.22 to 0.36 +/- 0.40%; P = 0.96). Plasma ET-1 levels were inversely correlated with the response of urinary osmolality after dDAVP administration (r = -0.62; P = 0.008).. Normoalbuminuric type 1 diabetic patients with DR have impaired renal response to dDAVP that is related to plasma ET-1 levels. Further studies are required to elucidate whether this tubular resistance to dDAVP might favor dehydration in these patients.

Topics: Adult; Albuminuria; Creatinine; Deamino Arginine Vasopressin; Diabetes Mellitus, Type 1; Diabetic Retinopathy; Endothelin-1; Female; Humans; Kidney Tubules; Male; Middle Aged; Osmolar Concentration; Young Adult

A high blood concentration of endothelin (ET)-1 may participate in the onset and progress of diabetic microangiopathy, resulting in neuropathy. We examined the therapeutic effects of prostaglandin E1 (PGE1), which possesses both a peripheral vasodilating action and inhibition of platelet aggregation, on diabetic microangiopathy. Increases in both skin temperature and peripheral never conduction velocity in diabetic patients were recorded four weeks after Lipo PGE1 administration. A quantitative decrease in urinary albumin concentration was also observed, suggesting its efficacy of action was on diabetic nephropathy. Lipo PGE1 administration reduced the elevated circulating plasma ET-1 levels in the diabetic patients. As an increase in ET-1 concentrations is thought to correlate with the onset and progress of diabetic microangiopathy, the reduction of plasma ET-1 concentration by Lipo PGE1 administration may be one reason for the improvement in diabetic neuropathy and nephropathy.

Topics: Adult; Aged; Aged, 80 and over; Albumins; Alprostadil; Angiotensins; Blood Glucose; Collagen Type IV; Cyclic AMP; Diabetic Angiopathies; Diabetic Nephropathies; Diabetic Neuropathies; Diabetic Retinopathy; Electric Conductivity; Electrocardiography; Endothelin-1; Fasting; Female; Glycated Hemoglobin; Humans; Hypertension; Male; Middle Aged; Peptidyl-Dipeptidase A; Peripheral Nerves; Renin; Skin Temperature

Microvascular alterations, impairment of coagulation, ischemia and diffuse endothelial damage are related to the progression of diabetic retinopathy. Defibrotide has been demonstrated to produce profibrinolytic, cytoprotective and vasofacilatory activities. The aim of the present study was to evaluate the therapeutic effect of Defibrotide in the treatment of nonproliferative diabetic retinopathy.. Two randomized age- and sex-matched groups (cases and controls) of 35 NIDDM patients presenting non-proliferative diabetic retinopathy were included in this study: cases were treated with Defibrotide (800-1600 mg daily) for two years.. All tested parameters (ETDRS visual acuity; computerized perimetry; retinography; fluorescein angiography), improved significantly (p<0.001) in Defibrotide-treated patients compared to controls. In our opinion, Defibrotide's manifold effects on vascular endothelia may account for this improvement by stimulation of tPA, PGI2, PGE2, thrombomodulin and modulation of endothelin-1 release.. Our preliminary data seem to suggest that Defibrotide could be proposed for medical treatment of nonproliferative diabetic retinopathy.

Topics: Aged; Case-Control Studies; Diabetes Mellitus, Type 2; Diabetic Retinopathy; Dinoprostone; Endothelin-1; Endothelium, Vascular; Epoprostenol; Female; Fibrinolytic Agents; Fluorescein Angiography; Follow-Up Studies; Fundus Oculi; Humans; Male; Middle Aged; Pilot Projects; Polydeoxyribonucleotides; Retina; Thrombomodulin; Tissue Plasminogen Activator; Treatment Outcome; Visual Field Tests

Endothelin-1 (ET-1) is a potent vasoconstrictor which seems to be involved in the pathogenesis of diabetic retinopathy (DR). However, studies on intraocular ET-1 in DR have been limited. Thus, we investigated aqueous ET-1 levels in patients with DR.. A total 85 subjects were included in this prospective study. Those were classified into three groups: advanced DR group included those with severe nonproliferative DR or proliferative DR, whereas early DR group included those with mild nonproliferative DR or moderative nonproliferative DR. Those who underwent cataract surgery and had no concomitant ocular disease were included in the control group. Aqueous humor levels of ET-1 were obtained before intravitreal bevacizumab injection (IVB) and after 1 month for the DR patients, and at the time of cataract surgery for the control group.. Advanced DR group included 40 eyes (47.1%), whereas early DR group did 19 eyes (22.4%), and control group (26 eyes, 30.5%). Mean aqueous ET-1 level was 10.1±4.1 pg/mL (6.0-21.0 pg/mL) in advanced DR group, 1.9±0.7 pg/mL (0.6-2.8 pg/mL) in early DR group, and 2.1±1.0 pg/mL (0.7-3.9 pg/mL) in control group (P < 0.001). Advanced DR group was further subdivided into severe nonproliferative DR (15 eyes, 12.8%) and proliferative DR (25 eyes, 34.3%). Mean aqueous ET-1 level was 10.1±4.3 pg/mL (6.0-20.1 pg/mL) in patients with severe nonproliferative DR, and 10.0±4.0 pg/mL (6.0-21.0 pg/mL) in those with proliferative DR (P = 0.928) at baseline. Mean ET-1 level at 1 month after intravitreal injection was 2.5±1.0 pg/mL (0.3-4.8 pg/mL) in patients with severe proliferative DR and 2.9±1.7 pg/mL (1.0-7.0 pg/mL) in those with proliferative DR (P = 0.443). Mean aqueous ET-1 level was significantly reduced in both groups (P < 0.001, respectively).. The mean aqueous ET-1 level was significantly higher in the eyes with advanced DR than those with early DR and the control group. The mean aqueous ET-1 level was significantly reduced after intravitreal injections in the advanced DR group. Based on our results, future studies on the exact role of ET-1 in the pathogenesis of DR and future implication for intervention would be helpful for managing DR.

Topics: Aqueous Humor; Bevacizumab; Cataract; Diabetes Mellitus; Diabetic Retinopathy; Endothelin-1; Humans; Prospective Studies

Diabetes elevates endothelin-1 (ET-1) in the vitreous and enhances constriction of retinal venules to this peptide. However, mechanisms contributing to ET-1-induced constriction of retinal venules are incompletely understood. We examined roles of sodium-hydrogen exchanger 1 (NHE1), protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), and extracellular calcium (Ca

Topics: Animals; Calcium; Diabetes Mellitus, Experimental; Diabetic Angiopathies; Diabetic Retinopathy; Endothelin-1; Imidazoles; Male; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Pyridines; Retinal Vein; Signal Transduction; Sodium-Hydrogen Exchanger 1; Swine; Vasoconstriction

The endothelin system (ES) plays a complex role in the pathogenesis of various eye diseases as a local regulator of vascular tone as well as many other physiological processes. Components of ES - endothelins and their receptors - can be found nearly in all cellular structures of the eye, their concentration increases in the presence of many eye diseases. In glaucoma, ES is involved in the mechanisms of eye hypertension by influencing the secretion and outflow of aqueous humor. The increase of endothelin level leads to the decrease of perfusion pressure, hypoxia, astrocyte proliferation, increase of density and rigidity of lamina cribrosa, apoptosis of neural cells, and has a profibrogenic effect. In retinal pathology, increase of endothelins disturbs autoregulation of retinal blood vessels changing the neurovascular interactions, breaks intercellular contacts in the retina, promotes neoangiogenesis. In diabetic retinopathy, ES contributes to the development of microangiopathy and proliferative vitreoretinopathy. The review discusses the possibility of correcting ES activity in the eye with medications by influencing its synthesis, cleavage and receptor binding.. Эндотелиновая система (ЭС) как локальный регулятор не только сосудистого тонуса, но многих других физиологических процессов играет многогранную роль в патогенезе целого ряда глазных болезней. Компоненты ЭС - эндотелины и их рецепторы - обнаружены практически во всех клеточных структурах глаза, и их содержание возрастает при многих глазных болезнях. При глаукоме ЭС участвует в механизмах повышения уровня внутриглазного давления (ВГД) путем влияния как на отток, так и на образование внутриглазной жидкости. Увеличение содержания эндотелина приводит к снижению перфузионного давления, гипоксии, пролиферации астроцитов, повышению плотности и ригидности решетчатой пластинки, апоптозу нервных клеток, оказывает профиброгенное действие. При патологии сетчатки увеличение содержания эндотелина нарушает процессы ауторегуляции сосудов сетчатки путем влияния на нейроваскулярные взаимодействия, нарушает межклеточные контакты в сетчатке, способствует ангиогенезу. При диабетической ретинопатии ЭС участвует в развитии микроангиопатий и пролиферативной витреоретинопатии. Обсуждается возможность медикаментозной коррекции активности ЭС в глазу путем воздействия на синтез, распад и взаимодействие эндотелинов с различными рецепторами.

Topics: Diabetic Retinopathy; Endothelin-1; Endothelins; Glaucoma; Humans; Retina; Retinal Vessels

To characterize the relationship between endothelin-1 and fibrosis in epiretinal membranes in proliferative diabetic retinopathy and explore the role of endothelial-mesenchymal transition in these membranes.. Membranes were obtained from eyes undergoing pars plana vitrectomy for complicated proliferative diabetic retinopathy or idiopathic epiretinal membrane. Through standard immunohistochemical techniques, we labeled membranes to explore the distribution of endothelin-1 and endothelin receptor B, comparing proliferative diabetic retinopathy and idiopathic epiretinal membranes. In addition, membranes were also labeled with markers for fibroblasts, endothelial, and glial cells and studied with confocal laser scanning microscopy. The intensity of endothelin-1 labeling was quantified using standard image analysis software.. Fourteen membranes were included in the analysis, nine from eyes with proliferative diabetic retinopathy and five idiopathic membranes. Flatmount diabetic membranes showed co-localization of endothelin-1 with S100A4 and CD31. Immunohistochemistry and quantitative analysis of cross-sectional membranes showed significantly higher endothelin-1 labeling in proliferative diabetic retinopathy membranes compared to idiopathic membranes (p<0.05). Diabetic membranes showed more elements staining positive for S100A4 compared to idiopathic membranes.. Epiretinal membrane formation in proliferative diabetic retinopathy involves higher tissue levels of endothelin-1 and fibroblastic activity. Furthermore, endothelin-1, endothelial and fibroblastic staining appear to be correlated, suggestive of endothelial-to-mesenchymal transition in proliferative diabetic retinopathy.

Topics: Adult; Cell Proliferation; Diabetic Retinopathy; Endothelin-1; Female; Fibrosis; Glial Fibrillary Acidic Protein; Humans; Male; Membranes; Middle Aged; Platelet Endothelial Cell Adhesion Molecule-1; Retina

Hypertension is an independent risk factor for diabetic retinopathy, yet anti-hypertensive medications such as blockade of angiotensin II do not completely protect against vision-threatening vascular disease. We hypothesized that the potent vasoactive factor, endothelin (ET), is up-regulated in diabetic retinopathy and antagonism of the ET type A receptor (ETRA) or ET type B receptor (ETRB) ameliorates retinal vascular leakage independently of any blood pressure lowering effects. Spontaneously hypertensive rats (SHR) and their normotensive and genetic controls, Wistar Kyoto rats, were randomized to become diabetic or non-diabetic and studied for 8 weeks. Rats were further randomized to receive by intravitreal injection the ETRA antagonist, BQ123, the ETRB antagonist, BQ788, or vehicle, 5 days after the induction of streptozotocin diabetes and 4 weeks later. The treatments had no effect on systolic blood pressure which remained elevated in SHR. ET-1, ET-2, ETRA and ETRB were expressed in retina and retinal pigment epithelium (RPE)/choroid and increased by hypertension or diabetes. BQ123 reduced ET-1 and ET-2 expression in retina and RPE/choroid, while BQ788 had a similar effect but did not influence the mRNA levels of ET-1 in retina. Retinal vascular leakage and Müller cell stress as well as vascular endothelial growth factor (VEGF) expression in retina and RPE/choroid, were increased by hypertension or diabetes and there was an additive effect of these conditions. Treatment with BQ123 or BQ788 effectively reduced these events as well as the elevated levels of inflammatory factors in the retina. Our findings indicate that local ET systems exist in the retina and RPE/choroid that are up-regulated by hypertension and diabetes. The ability of locally delivered ET receptor antagonists to supress these overactive ET systems and reduce retinal vascular leakage and VEGF in the presence of hypertension indicate the potential of these approaches for the treatment of diabetic retinopathy.

Topics: Animals; Antihypertensive Agents; Blood Pressure; Blood-Retinal Barrier; Choroid; Diabetes Mellitus, Experimental; Diabetic Retinopathy; Endothelin A Receptor Antagonists; Endothelin B Receptor Antagonists; Endothelin-1; Endothelin-2; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique, Indirect; Intravitreal Injections; Ocular Hypertension; Oligopeptides; Peptides, Cyclic; Piperidines; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Real-Time Polymerase Chain Reaction; Retina; Retinal Pigment Epithelium; Streptozocin

Topics: Administration, Topical; Animals; Apoptosis; Bosentan; Diabetes Mellitus, Experimental; Diabetic Retinopathy; Endothelin-1; Female; Glial Fibrillary Acidic Protein; Humans; Immunohistochemistry; In Situ Nick-End Labeling; In Vitro Techniques; Male; Rabbits

PurposeThe purpose of this study was to determine the association between aqueous ET-1 levels and total retinal blood flow (TRBF) in patients with non-insulin-dependent type 2 diabetes mellitus (T2DM) and early non-proliferative diabetic retinopathy (NPDR).Patients and methodsA total of 15 age-matched controls and 15 T2DM patients with NPDR were recruited into the study. Aqueous humor (~80-120 μl) was collected before cataract surgery to measure the levels of ET-1 using suspension multiplex array technology. Four weeks post surgery, six images were acquired to assess TRBF using the prototype RTVue Doppler FD-OCT (Optovue, Inc., Fremont, CA, USA) with a double circular scan protocol. At the same visit, forearm blood was collected to determine plasma glycosylated hemoglobin (A1c) levels.ResultsAqueous ET-1 was significantly elevated in the NPDR group compared with the control group (3.5±1.8 vs 2.2±0.8, P=0.02). TRBF was found to be significantly reduced in the NPDR group compared with the control group (34.5±9.1 vs 44.1±4.6 μl/min, P=0.002). TRBF and aqueous ET-1 were not correlated within the NPDR group (r=-0.24, P=0.22). In a multivariate analysis, high A1c was associated with reduced TRBF and aqueous ET-1 levels across control and NPDR groups (P<0.01).ConclusionAqueous ET-1 levels were increased while TRBF was reduced in patients with NPDR compared with the control group. Although not directly associated, the vasoconstrictory effects of ET-1 are consistent with a reduced TRBF observed in early DR. ET-1 dysregulation may contribute to a reduction in retinal blood flow during early DR.

Topics: Aged; Aqueous Humor; Biomarkers; Diabetes Mellitus, Type 2; Diabetic Retinopathy; Endothelin-1; Female; Humans; Male; Regional Blood Flow; Retinal Vessels; Tomography, Optical Coherence

To assess vitreous and plasma changes of vascular endothelial growth factor A (VEGF-A), adrenomedullin (ADM) and endothelin-1 (ET-1) in proliferative diabetic retinopathy (PDR).. 9 patients with PDR in type 2 diabetes (T2DM) and 11 age-matched non-diabetic patients were enrolled. The levels of VEGF-A, ADM and ET-1 were measured using an enzyme (ELISA) and a radioimmunoassay (RIA) both in vitreous and plasma samples.. Vitreous ADM and VEGF-A levels were significantly higher in PDR patients (p=0.04 and p=0.02), whereas no differences were found in ET-1 levels (p=0.29). Plasma ADM levels were significantly higher in the PDR group (p<0.01), whereas no significant differences were found in the plasma ET-1 and VEGF-A levels (p=0.30 and p=0.37). The ADM vitreous/plasma ratio was significantly reduced in PDR group.. The role of ET-1 in advanced PDR is still controversial; it has been supposed a role limited to induce hypoxic state and promote angiogenesis in the early phases. Once the neo-angiogenic process starts, other mediators are mainly involved as VEGF and ADM. Our findings suggest that ADM is an important marker of advanced PDR as well as VEGF. Conversely, ET-1 is not significantly involved in the advanced stage of PDR.

Topics: Adrenomedullin; Adult; Aged; Aged, 80 and over; Case-Control Studies; Diabetes Mellitus, Type 2; Diabetic Retinopathy; Endothelin-1; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Female; Humans; Male; Middle Aged; Vascular Endothelial Growth Factor A; Vitreous Body

Fufang Xueshuantong Capsule, an herbal formula licensed for clinical use in China, which is composed of Panax notoginseng (Burkill) F.H. Chen, Salvia miltiorrhiza Bunge, Astragalus membranaceus (Fisch.) Bunge, and Scrophularia ningpoensis Hemsl, has proven effective for the treatment of diabetic retinopathy. However, its bioactive constituents are still ambiguous. In this study, the therapeutic effects of a combination of the main constituents of Fufang Xueshuantong Capsule (cFXT) were evaluated in streptozotocin (STZ)-induced retinal lesions to identify the bioactive constituents.. Sprague-Dawley rats, except for those in the control group (vehicle+vehicle), were administered a single injection of 60mg/kg STZ. One-week later, STZ-treated rats were randomly divided into three groups-one STZ group (STZ+vehicle) and two cFXT treatment groups (STZ+cFXT). The rats in the latter two groups received cFXT 44.8mg/kg or cFXT 22.4mg/kg by intragastric gavage once per day, for 24 consecutive weeks. The rats in the control and STZ groups received the vehicle in the same way. Body weights and fasting blood glucose levels were recorded every four weeks. After treatment, hemorheological tests were performed to record the erythrocyte aggregation indexes, blood viscosity, and plasma viscosity. The trypsin digestion method was used to observe pericyte and acellular capillary counts in the retina. Ultraviolet spectrophotometry was utilized to measure the activity of aldose reductase (AR) by measuring the nicotinamide adenine dinucleotide phosphate (NADPH) consumption at 340nm. An immunohistochemical assay was used to observe the expressions of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) in the retina. The expression levels of intercellular adhesion molecule-1 (ICAM-1), endothelin-1 (RT-1),and occludin in the retina were tested by the western blot assay.. cFXT is composed of 991.44mg/g saponins of Panax notoginseng, 1.62mg/g harpagoside, 0.70mg/g cryptotanshinone, 0.74mg/g tanshinone I, and 5.50mg/g astragaloside A. Although it showed no effects on the increased body weight and blood glucose levels induced by STZ in rats. However, it showed a tendency to attenuate the increase in erythrocyte aggregation, plasma viscosity, and acellular vessel and pericyte loss, paralleled with a reversal of the hyper-activation of AR, the hyper-expression of VEGF, ICAM-1, and ET-1, and the hypo-expression of PEDF and occludin in the retinas of STZ-treated rats.. The saponins of Panax notoginseng, harpagoside, cryptotanshinone, tanshinone I, and astragaloside A are the main bioactive constituents of Fufang Xueshuantong Capsule and contribute to the attenuation of STZ-induced retinal lesions in rats. These constituents can be used as the base to optimize a new drug for the treatment of diabetic retinopathy, and can be selected for quality control of Fufang Xueshuantong Capsules.

Topics: Animals; Blood Glucose; Capsules; Diabetes Mellitus, Experimental; Diabetic Retinopathy; Drugs, Chinese Herbal; Endothelin-1; Eye Proteins; Hypoglycemic Agents; Intercellular Adhesion Molecule-1; Male; Nerve Growth Factors; Occludin; Phytotherapy; Rats; Rats, Sprague-Dawley; Retina; Saponins; Serpins; Streptozocin; Vascular Endothelial Growth Factor A

To examine the relationships of serum markers of inflammation and endothelial function to diabetic retinopathy.. We recruited 224 patients with diabetes (85 with Type 1 and 139 with Type 2 diabetes) aged 18-70 years. Serum markers of inflammation (high-sensitivity C-reactive protein) and endothelial function (soluble intercell adhesion molecule-1, vascular cell adhesion molecule-1, E-selectin, endothelin-1 and total nitrite) were assessed using nephelometry, immunoassays and spectroscopy. Diabetic retinopathy was graded from two-field fundus photographs according to the Airlie House Classification system and was categorized into no diabetic retinopathy, mild non-proliferative diabetic retinopathy, moderate non-proliferative diabetic retinopathy and vision-threatening diabetic retinopathy, the latter comprising severe non-proliferative diabetic retinopathy, proliferative diabetic retinopathy or clinically significant macular oedema. Multinomial logistic regression was used to assess the associations between serum markers and diabetic retinopathy.. In the study, 64% of patients (144/224) had diabetic retinopathy and 25% (57/244) had vision-threatening diabetic retinopathy. After controlling for age, gender, diabetes duration, HbA1c , systolic blood pressure, total and HDL cholesterol, smoking, the use of insulin or oral hypoglycaemic agents, nephropathy and cardiovascular disease, a positive association was found between increasing high-sensitivity C-reactive protein levels and the presence of vision-threatening diabetic retinopathy (odds ratio 1.26; 95% CI 1.05-1.51, per sd increase in high-sensitivity C-reactive protein). After stratifying by BMI ( ≥ 30 and < 30 kg/m(2) ), this association was found to be more pronounced in people with a BMI ≥ 30 kg/m(2) (odds ratio 2.9; P for interaction = 0.019). No associations were found between serum markers of endothelial activation and diabetic retinopathy.. Higher C-reactive protein levels, but not markers of endothelial function, may be related to more severe diabetic retinopathy. This finding suggests that inflammatory processes are involved in severe diabetic retinopathy, particularly in patients with a BMI ≥ 30 kg/m(2) .

Topics: Adolescent; Adult; Aged; Biomarkers; C-Reactive Protein; Cross-Sectional Studies; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diabetic Retinopathy; E-Selectin; Endothelin-1; Endothelium, Vascular; Female; Humans; Inflammation; Intercellular Adhesion Molecule-1; Logistic Models; Male; Middle Aged; Nitrates; Ophthalmoscopes; Severity of Illness Index; Vascular Cell Adhesion Molecule-1; Young Adult

We sought to determine the effects of atrasentan, a selective endothelin-A receptor antagonist, on the retinal vascular and structural integrity in a db/db mouse, an animal model of type 2 diabetes and diabetic retinopathy.. Diabetic mice, 23 weeks old, were given either atrasentan or vehicle treatment in drinking water for 8 weeks. At the end of the treatment period, eyes underwent trypsin digest to assess the retinal vascular pathology focusing on capillary degeneration, endothelial cell, and pericyte loss. Paraffin-embedded retinal cross sections were used to evaluate retinal sublayer thickness both near the optic nerve and in the retinal periphery. Immunohistochemistry and TUNEL assay were done to evaluate retinal cellular and vascular apoptosis.. Compared with untreated db/db mice, atrasentan treatment was able to ameliorate the retinal vascular pathology by reducing pericyte loss (29.2% ± 0.4% vs. 44.4% ± 2.0%, respectively, P < 0.05) and capillary degeneration as determined by the percentage of acellular capillaries (8.6% ± 0.3% vs. 3.3% ± 0.41%, respectively, P < 0.05). A reduction in inner retinal thinning both at the optic nerve and at the periphery in treated diabetic mice was also observed in db/db mice treated with atrasentan as compared with untreated db/db mice (P < 0.05). TUNEL assay suggested that atrasentan may decrease enhanced apoptosis in neuroretinal layers and vascular pericytes in the db/db mice.. Endothelin-A receptor blockade using atrasentan significantly reduces the vascular and neuroretinal complications in diabetic mice. Endothelin-A receptor blockade is a promising therapeutic target in diabetic retinopathy.

Topics: Animals; Apoptosis; Atrasentan; Cell Count; Diabetes Mellitus, Experimental; Diabetic Retinopathy; Endothelin Receptor Antagonists; Endothelin-1; Female; Immunohistochemistry; In Situ Nick-End Labeling; Mice; Mice, Inbred C57BL; Pyrrolidines; Retinal Ganglion Cells; Retinal Vessels

Ocular ischemic microenvironment plays a critical role in the progression of diabetic retinopathy (DR). In this study, we investigated the effect of vitreous and aqueous obtained from proliferative DR patients on the function of CD34⁺ cells derived from healthy humans. Human CD34⁺ cells were incubated with vitreous or aqueous of subjects with PDR. After incubation, cell migration of CD34⁺ was evaluated with CXCL12. Intracellular levels of nitric oxide (NO) were measured with DAF-FM. Tube formation assay was used to evaluate the effect of treated CD34⁺ cells on in vitro angiogenesis. Angiogenic protein array and mass spectrometry (MS) were performed to ascertain the factors secreted by healthy nondiabetic CD34⁺ cells exposed to diabetic vitreous or aqueous. PDR vitreous/aqueous reduced migration of CD34⁺ cells (672.45 ± 42.1/736.75 ± 101.7 AFU; P < 0.01) and attenuated intracellular NO levels (182 ± 1.4/184.5 ± 6.3 AFU, P = 0.002). Pretreatment with PDR vitreous suppressed tube formation of human retinal endothelial cells (64 ± 1.6 vs. 80 ± 2.5). CD34⁺ exposed to PDR vitreous resulted in the increased expression of CXCL4 and serpin F1, whereas CD34⁺ exposed to PDR aqueous showed increased expression of CXCL4, serpin F1, and endothelin-1 (ET-1). MS analysis of CD34⁺ (exposed to PDR vitreous) expressed J56 gene segment, isoform 2 of SPARC-related modular calcium-binding protein 2, isoform 1 of uncharacterized protein c1 orf167, integrin α-M, and 40s ribosomal protein s21. Exposure of healthy nondiabetic CD34⁺ cells to PDR vitreous and aqueous resulted in decreased migration, reduced generation of NO, and altered paracrine secretory function. Our results suggest that the contribution of CD34⁺ cells to the aberrant neovascularization observed in PDR is driven more by the proangiogenic effects of the retinal cells rather than the influence of the vitreous.

Topics: Adult; Adult Stem Cells; Aged; Antigens, CD34; Aqueous Humor; Bone Marrow Cells; Cells, Cultured; Chemotaxis; Diabetic Retinopathy; Endothelin-1; Eye Proteins; Humans; Middle Aged; Neovascularization, Pathologic; Nerve Growth Factors; Nitric Oxide; Peptide Mapping; Platelet Factor 4; Retina; Serpins; Vitreoretinopathy, Proliferative; Vitreous Body

To evaluate the values of ocular hemodynamics and serum endothelin-1 (ET-1) in the early diagnosis of diabetic retinopathy (DRP).. A total of 85 DRP patients were examined by ophthalmoscope and fluorescein angiography and divided into 3 groups: no obvious retinopathy (n = 20), non-proliferative diabetic retinopathy (n = 35) and proliferative diabetic retinopathy (n = 30). Control group included 15 healthy volunteers. The peak systolic velocity (PSV), end diastolic velocity (EDV) and resistance index (RI) of ophthalmic artery (OA) and central retinal artery (CRA) were measured by color Doppler energy imaging. The level of endothelin-1 was measured by radioimmunoassay. Then plasma ET-1 levels and eye vascular hemodynamic parameters were compared with correlation and diagnostic specificity after data analysis.. Compared with the control group, DRP, EDV and PSV decreased progressively while RI increased gradually (P < 0.05). EDV and RI were more sensitive than PSV in diagnosis. The plasma level of ET-1 was significantly higher than that of control group (P < 0.05). And it was correlated positively with RI and negatively with PSV and EDV of CRA. The diagnostic value of EDV was higher than that of ET-1 level.. Ocular hemodynamics become abnormal during the early stage of DRP and worsen with the progress of DRP. An elevated plasma level of ET-1 may lead to the abnormal of retinal hemodynamics. The test of plasma ET-1 and the examination of ocular hemodynamics may play an important role in the early diagnosis of DRP.

Topics: Adult; Aged; Case-Control Studies; Diabetic Retinopathy; Disease Progression; Endothelin-1; Female; Hemodynamics; Humans; Male; Middle Aged; Ultrasonography, Doppler, Color

Endothelial dysfunction plays an important role in the development of diabetic retinopathy. The aim of this study was to evaluate endothelial dysfunction using different approaches in patients with type 1 diabete mellitus with early stages of diabetic retinopathy. For this purpose, we investigated the serum levels of cellular adhesion molecules, including intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1) and endothelin-1 (ET-1), which have emerged as specific markers of endothelial dysfunction, and measured the flow-mediated dilatation (FMD), a noninvasive technique used to evaluate endothelial dysfunction.. The study group included 59 patients with type 1 diabetes mellitus (DM) and 30 age-matched healthy control subjects. The diabetic patients were divided into two groups according to the ophthalmoscopic findings: Group 1, composed of type 1 diabetic patients having no signs of diabetic retinopathy (DRP), and Group 2, composed of type 1 diabetic patients having findings of the early stages of nonproliferative diabetic retinopathy (NPDR).. The serum levels of ET-1 (fmol/mL), ICAM-1 (ng/mL) and VCAM-1 (ng/mL) were 8.52±0.699 vs. 478.39±46.22 vs. 728.64±35.081 in the patients without retinopathy, 8.91±1.354 vs. 451.79±48.262 vs. 863.59±62.37 in the diabetic patients with NPDR and 10.73±1.04 vs. 608.15±74.92 vs. 872.95±57.63 in the control group. There were no significant differences in the serum levels of the three molecules between the groups. The FMD values were 6.51±0.46% in the diabetic patients without retinopathy, 6.66±0.29% in the diabetic patients with NPDR and 6.68±0.51% in the control group. No significant differences were found between the groups.. The early stages of diabetic retinopathy cannot be considered in the evaluation of systemic markers of endothelial dysfunction.

Topics: Adult; Biomarkers; Blood Flow Velocity; Diabetes Mellitus, Type 1; Diabetic Retinopathy; Endothelin-1; Female; Humans; Intercellular Adhesion Molecule-1; Male; Vascular Cell Adhesion Molecule-1; Vasodilation

Although hyperglycemia is implicated in retinal vascular dysfunction associated with the development of diabetic retinopathy, the temporal influence of hyperglycemia on retinal arteriolar reactivity remains unclear. Development of a large animal model of diabetes relevant to the human retina for evaluation of vascular function is also lacking. Herein, we examined nitric oxide (NO)-mediated dilation and endothelin-1 (ET-1)-induced constriction in retinal arterioles at various time periods in a porcine model of type 1 diabetes.. Retinal arterioles were isolated from streptozocin-induced diabetic pigs (2, 6, and 12 weeks of hyperglycemia, 427 ± 23 mg/dL) and age-matched control pigs (73 ± 4 mg/dL), and then cannulated and pressurized for vasoreactivity study using videomicroscopic techniques.. Retinal arterioles isolated from control and diabetic pigs developed comparable levels of myogenic tone. The endothelium-dependent NO-mediated vasodilations to bradykinin and stepwise increases in luminal flow were significantly reduced within 2 weeks of hyperglycemia. The inhibitory effect was comparable following 6 and 12 weeks of hyperglycemia. However, the endothelium-independent vasodilation to sodium nitroprusside was unaffected. Constriction of retinal arterioles to ET-1 was unaltered at all time periods of hyperglycemia.. Our findings provide the first direct evidence for selective impairment of endothelium-dependent NO-mediated dilation of retinal arterioles within 2 weeks of hyperglycemia in a pig model of diabetes. By contrast, the ability of arteriolar smooth muscle to dilate to NO donor or contract to ET-1 was unaffected throughout the study period. This endothelial vasodilator dysfunction during early diabetes may contribute to development of retinopathy with chronic hyperglycemia.

Topics: Animals; Arterioles; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetic Retinopathy; Dose-Response Relationship, Drug; Endothelin-1; Endothelium, Vascular; Male; Muscle, Smooth, Vascular; Nitric Oxide; Retinal Artery; Sus scrofa; Time Factors; Vasodilator Agents

To investigate the roles of endothelin-1(ET-1), TNF-alpha, IL-6 in the pathogenesis of proliferative diabetic retinopathy (PDR) in type 2 diabetes.. Vitreous and blood serum samples were collected during vitrectomy from 19 patients with PDR and 15 patients who underwent vitrectomy for other reasons. The concentrations of ET-1, TNF-alpha, IL-6, vWF, sE-selectin were determined by ELISA.. Intraocular and serous concentrations of ET-1, TNF-alpha, IL-6, vWF, sE-selectin were higher in patients with PDR than in the control group. The vitreous ET-1/plasma ET-1 ratios the group of diabetic patients and in the control group were similar. Also TNF-alpha, IL-6 vitreous/plasma ratio were not statistically different between the analysed groups. Correlation between intraocular ET-1 and TNF-alpha concentrations in patients with PDR and between the increases in both factors in the vitreous and HbA(1)c concentration were shown. In the vitreous the increase in vWF depended on elevated levels of vWF in the serum. E-selectin concentration correlated with diastolic blood pressure.. These data provide evidence of the activation of the local synthesis of ET-1, TNF-alpha, IL-6 in PDR. The relationship between the increase in vitreous ET-1, TNF-alpha concentrations and HbA(1)c concentration is a important confirmation of the necessity to optimise diabetes treatment.

Topics: Aged; Cytokines; Diabetic Retinopathy; E-Selectin; Endothelin-1; Female; Humans; Hypertension; Inflammation Mediators; Interleukin-6; Male; Middle Aged; Tumor Necrosis Factor-alpha; Vitreous Body; von Willebrand Factor

To (i) investigate expression of the endothelin-1 (ET-1) gene in peripheral blood mononuclear cells (PBMCs) of patients with type 2 diabetes mellitus (DM) and (ii) determine what correlations, if any, exist between expression of ET-1 in patients with type 2 DM and treatment, clinical features, and biochemical markers in diabetic retinopathy (DR).. The study group included 58 patients with type 2 DM, subdivided into three subgroups: those without DR (n=19), those with nonproliferative DR (NPDR; n=28), and those with proliferative DR (PDR; n=11). The control group consisted of 60 individuals. In all groups the mRNA level of ET-1 was estimated using real-time quantitative reverse transcription PCR.. The mRNA level of ET-1 in patients with NPDR was significantly higher than in those without DR. An increase in ET-1 expression was observed in patients with PDR as opposed to those without DR. Compared to controls, the mRNA level of ET-1 was significantly higher both in patients with NPDR and those with PDR. Duration of DM, insulin therapy, and serum creatinine levels were associated with increased mRNA level of ET-1, whereas medication with sulfonylurea or angiotensin-converting enzyme inhibitors had the opposite effect.. Expression of ET-1 in PBMCs may be associated with severity of DR in patients with type 2 DM. Long-standing clinical course of DR; medication with insulin, sulfonylurea, or ACE inhibitors; and serum creatinine levels are factors possibly associated with changes in ET-1 expression in PBMCs.

Topics: Adult; Aged; Aged, 80 and over; Case-Control Studies; Diabetes Mellitus, Type 2; Diabetic Retinopathy; Endothelin-1; Female; Gene Dosage; Gene Expression Regulation; Humans; Male; Middle Aged; RNA, Messenger

Hyperglycemia mediates endothelial cell dysfunction through a number of potential mechanisms that could result in the decrease of retinal blood flow early in diabetes. The aim of this study was to explore the role of endothelin receptor A (ET(A)) in the early decrease of retinal blood flow in diabetic mice. Diabetes was induced by streptozotocin, then ∼1 wk later the mice were administered drinking water with or without the ET(A) receptor antagonist atrasentan (7.5mg/kg/day) for the following 3 weeks. Non-diabetic age-matched mice with or without atrasentan were included as controls. For each mouse, measurements of retinal vascular diameters and red blood cell (RBC) velocities were obtained via intravital microscopy for the 5-7 feed arterioles (and draining venules) extending out of (and into) the optic disk, and from these values, flow rates and wall shear rates were calculated. Additionally, the number of retinal capillaries was counted by fluorescent immunostaining of platelet-endothelial cell adhesion molecule-1 (PECAM-1). Diabetes induced statistically significant decreases in RBC velocity, flow rate, and wall shear rate, with these alterations partially inhibited by atrasentan. No changes were observed in PECAM-1 expression among groups. The changes induced by diabetes, and the attenuation provided by atrasentan, were greater in the smaller retinal arterioles. In summary, ET(A) appears to play a role in the early decreases in retinal blood flow in a mouse model of diabetes.

Topics: Administration, Oral; Animals; Atrasentan; Blood Flow Velocity; Blood Glucose; Body Weight; Diabetes Mellitus, Experimental; Diabetic Retinopathy; Endothelin A Receptor Antagonists; Endothelin-1; Fluorescent Antibody Technique, Indirect; Male; Mice; Mice, Inbred C57BL; Platelet Endothelial Cell Adhesion Molecule-1; Pyrrolidines; Regional Blood Flow; Retinal Vessels

We tested the hypothesis that genetic variants in vasoactive and angiogenic factors regulating the retina vasculature contribute to the development of diabetic retinopathy (DR).. A case-control study was performed to study the genetic association between DR and polymorphic variants of EDN1 (Lys198Asn), LTA (IVS1-80C>A, IVS1-206G>C, IVS1-252A>G), eNOS (Glu298Asp), and ITGA2 (BgI II) in a Chinese population with type 2 diabetes mellitus. A well defined population with type 2 diabetes, consisting of 127 controls and 216 DR patients, was recruited.. A higher frequency of the Asn/Asn genotype of EDN1 was found in individuals with at least 10 years of diabetes and no retinopathy (controls) compared with DR patients with any duration of diabetes (DR: 2.3%; control: 11.0%; p=0.0002). The Asn allele was also more frequent in controls than DR patients (DR: 16.4%; control: 29.5%; p=0.007). Multiple logistic regression analysis showed that the Asn/Asn genotype was the factor most significantly associated with reduced risk of DR (odds ratio=0.19; 95% CI: 0.07-0.53; p=0.002) and with late onset of diabetes (Asn/Asn: 59 years; Lys/Lys + Lys/Asn: 53 years; p=0.02). Moreover, the Lys/Lys genotype was more common among patients with nonproliferative (75.7%) than proliferative DR (56.9%; p=0.008). The distributions of Lys198Asn alleles in hypertension did not differ from normotensive subjects. No associations between DR and polymorphisms of LTA, eNOS, or ITGA2 were detected, and there were no detectable gene-gene or gene-environmental interactions among the polymorphisms.. The Asn/Asn genotype of EDN1 was associated with a reduced risk of DR and with delayed onset of type 2 diabetes.

Topics: Age of Onset; Aged; Asparagine; Case-Control Studies; China; Diabetes Mellitus, Type 2; Diabetic Retinopathy; Endothelin-1; Female; Gene Frequency; Genetic Predisposition to Disease; Genotype; Humans; Hypertension; Lysine; Male; Middle Aged; Odds Ratio; Polymorphism, Single Nucleotide; Regression Analysis

There are changes in blood flow during the clinical stages of diabetic retinopathy with increasing leukostasis and secondary elaboration of cytokines. This study evaluated the vitreous concentrations of haemodynamic-related (endothelin-1 (ET-1) and nitric oxide (NO)), inflammatory and anti-inflammatory (interleukin-1 receptor antagonist, IL-1 Ra) cytokines in the diabetic patients (with nonproliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR)), compared them with those of control patients (full thickness macular hole, FTMH) and correlated to macular structural indices.. Vitreous samples from five FTMH patients representing normal controls were analysed together with the vitreous samples of 15 patients with NPDR and five with PDR. The vitreous concentrations of nitrite (total NO), ET-1, and prostacyclin was determined using ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. A sandwich luminescent immunoassay technique was used to determine IL-1beta and IL-1 Ra concentrations.. In the different clinical groups, there were no differences in the vitreous NO and prostacyclin concentrations. In NPDR, the median ET-1 concentration (0.7 pg/ml SD +/-0.8 pg/ml) was significantly reduced (P<0.05), compared to PDR (6.35 pg/ml SD +/-0.6 pg/ml) and FTMH (3.6 pg/ml SD +/-0.14 pg/ml). Its concentration also positively correlated with foveal thickness and macular volume (P<0.05) in patients with NPDR and macular oedema. IL-1 beta was detected in PDR, and diabetic patients demonstrated a lower concentration of the anti-inflammatory cytokine IL-1 Ra.. Reduced concentrations of ET-1 in NPDR may reflect the haemodynamic changes of NPDR. The IL-1 Ra concentration suggests a change in the anti-inflammatory environment of the diabetic retina.

Topics: Adult; Aged; Cytokines; Diabetes Mellitus, Type 2; Diabetic Retinopathy; Endothelin-1; Enzyme-Linked Immunosorbent Assay; Epoprostenol; Humans; Inflammation Mediators; Interleukin-1beta; Macular Edema; Middle Aged; Nitric Oxide; Receptors, Interleukin-1; Vitreous Body

To evaluate the relationship between oxidative stress and endothelial dysfunction (ED) in diabetic patients without clinical macrovascular complications.. In 27 type 1, 56 type 2 diabetic patients and 35 healthy controls the redox state (GSH, GSSG; enzymatic method), endothelin-1 (ET-1; ELISA) and von Willebrand factor (vWF; ELISA) plasma levels, urinary vascular endothelial growth factor (VEGF; ELISA) were measured.. Decreased GSH levels (p<0.05, type 1 and type 2), GSH/GSSG ratio (p<0.05 type 1, p<0.001 type 2) and elevated vWF levels (p<0.001, type 1 and type 2) were observed in diabetic patients in comparison with controls. A negative correlation between GSH and vWF (p<0.02 and p<0.001, in type 1 and type 2, respectively) and GSH and BMI (p<0.02 in type 1 and type 2) was observed. ET-1 was positively correlated to age (p<0.05) and diabetes duration (p<0.03) in type 1, while vWF was correlated to systolic blood pressure (p<0.05) in type 2 diabetic patients. Urinary VEGF was higher in type 2 (p<0.05) in comparison with type 1 diabetic patients and was correlated to glycemia (p<0.05) and systolic blood pressure (p<0.05).. These data might indicate that markers of oxidative stress and ED are altered in diabetic patients without clinical macrovascular complications.

Topics: Adult; Biomarkers; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diabetic Retinopathy; Endothelin-1; Endothelium, Vascular; Female; Glutathione; Humans; Male; Middle Aged; Oxidative Stress; Reference Values; Vascular Endothelial Growth Factor A; von Willebrand Factor

To evaluate the relationship of plasma endothelin-1 (ET-1) levels, a marker of endothelial dysfunction, and urinary albumin excretion in patients with type 2 diabetes mellitus (DM).. Cross-sectional study was conducted in 279 patients (132 males, mean age: 58.7+/-11.0 years, mean DM duration: 11.3+/-8.1 years). Urinary albumin excretion, ET-1, and insulin were measured. Insulin sensitivity was estimated by homeostasis model assessment (HOMA-ir) index.. ET-1 was associated with urinary albumin excretion after controlling for age, gender, body mass index, blood pressure, HbA1c test, and total cholesterol (R=0.436; adjusted R(2)=0.190, P<0.01). Furthermore, there was a progressive increase in plasma ET-1 levels from patients with normoalbuminuria (n=187, 0.92+/-0.50pg/ml), microalbuminuria (n=68, 1.13+/-0.52pg/ml) to macroalbuminuria (n=24, 1.93+/-1.10pg/ml, P<0.01).. There is an independent association of plasma ET-1 levels with urinary albumin excretion. In addition, plasma ET-1 levels started to increase in the normal values of urinary albumin excretion suggesting that in patients with type 2 DM endothelial dysfunction is already present, in urinary albumin excretion values considered normal.

Topics: Age of Onset; Aged; Albuminuria; Cross-Sectional Studies; Diabetes Mellitus, Type 2; Diabetic Angiopathies; Diabetic Retinopathy; Endothelin-1; Endothelium, Vascular; Female; Humans; Male; Middle Aged

To investigate the potential role of endothelin-1 (ET-1), a potent vasoconstrictor with mitogenic properties, in the pathogenesis of proliferative diabetic retinopathy (PDR).. Plasma and vitreous samples were collected from normal patients (controls; n = 25), diabetic patients with PDR (n = 25), and diabetic patients with non-PDR (n = 25). The patients had to have epiretinal membranes (ERMs) or other ocular conditions that made them candidates for vitrectomy. Immunoreactive ET-1 (IR-ET-1) was assayed in plasma and vitreous samples by radioimmunoassay. IR-ET-1 was immunohistochemically localized in ERMs. Expression of endothelin receptors A (ETA) and B (ETB) was confirmed by reverse transcription-polymerase chain reaction analysis.. IR-ET-1 levels in plasma and vitreous samples from diabetic patients were higher (P < 0.0001) than those in samples from the control group. The levels for patients with PDR were even higher (P < 0.0001) than those for patients with non-PDR. Eyes with ERMs in the PDR group had the highest vitreous IR-ET-1 levels (14.67 +/- 0.67 pg/mL). IR-ET-1 was localized in the cellular and stromal components of ERMs in diabetic and nondiabetic patients. Furthermore, the ETA and ETB receptors were expressed in both diabetic and nondiabetic ERMs.. Diabetic patients with PDR and ERMs had the highest plasma and vitreous IR-ET-1 levels. ET-1 and its ETA and ETB receptors were present in ERMs. These data suggest that ET-1 is involved in diabetic vitreoretinal disease.

Topics: Aged; Aged, 80 and over; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diabetic Retinopathy; Endothelin-1; Epiretinal Membrane; Female; Humans; Immunoenzyme Techniques; Male; Middle Aged; Radioimmunoassay; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Vitreous Body

To investigate the diagnostic efficacy and clinical application value of plasma endothelin-1 for diabetic retinopathy using receiver operating characteristic (ROC) curve analysis.. This was a prospective investigational study. Funduscopy and fundus fluorescein angiography were used as gold standards for the diagnosis of diabetic retinopathy. Plasma endothelin-1 was measured in 96 diabetic patients with retinopathy (the case group) and 144 diabetic patients without retinopathy (the control group). Enumerative data were listed in a fourfold table. The measurement data were analyzed by Student's t test and evaluated by cross-table analysis and ROC curve analysis.. (1) The plasma endothelin-1 concentration was higher in the case group than the control group (p = 0.002 < 0.01). (2) If the plasma endothelin-1 level of 162 pg/ml was adopted as the threshold for clinical diagnosis of diabetic retinopathy, the diagnostic sensitivity was 71.2%, diagnostic specificity 58% and diagnostic accuracy 66%. The positive predictive value was 69.81% and the negative predictive value 59.46%, and the positive likelihood ratio was 1.69 and the negative likelihood ratio 0.50. (3) When plasma endothelin-1 was used as a diagnostic criterion for diabetic retinopathy, the area under the ROC curve was 0.737.. Plasma endothelin-1 plays an important role in the development and progression of diabetic retinopathy. When 162 pg/ml of plasma endothelin-1 was adopted as the diagnostic threshold, the diagnostic accuracy was medium; hence, the plasma endothelin-1 level can be used as the first step for diabetic retinopathy screening.

Topics: Aged; Diabetes Mellitus, Type 2; Diabetic Retinopathy; Endothelin-1; Female; Humans; Male; Middle Aged; Prospective Studies; ROC Curve

1. The aims of the present study were to examine whether: (i) upregulation of the endothelin (ET) pathway is involved in impairment of vascular relaxation and early retinopathy in diabetic rats; and (ii) vascular and retinal abnormalities respond to the total triterpene acid (TTA) isolated from Fructus Corni compared with responses to the novel endothelin receptor antagonist CPU0213 and aminoguanidine (AMG), a special antagonist for advanced glycation end-products (AGE) and inducible nitric oxide synthase (iNOS). 2. Male Sprague-Dawley rats were randomized into five groups, namely a normal control and four diabetic groups, which included an untreated diabetic group and groups treated with AMG (100 mg/kg, i.g.), CPU0213 (30 mg/kg, s.c.) or TTA (50 mg/kg, i.g.). Diabetes was induced by single injection of streptozotocin (60 mg/kg, i.p.) on the 1st day. The mRNA expression of prepro-endothelin-1 (ppET-1), endothelin-converting enzyme (ECE) and iNOS in the thoracic aorta and mRNA for ET(A) receptors and iNOS in the retina were detected by reverse transcription-polymerase chain reaction. Vasorelaxation to acetylcholine (ACh) and functional assessment of nitric oxide (NO) bioavailability was determined in the thoracic aorta. 3. We observed upregulated mRNA expression of iNOS, ppET-1 and ECE in the thoracic aorta and upregulated mRNA for the ET(A) receptor and iNOS in the retina in the untreated diabetic group. Vasodilatation mediated by ACh and NO bioavailability were markedly reduced in the thoracic aorta compared with the normal control group. These abnormalities were essentially reversed by TTA, CPU0213 or AMG, with the exception with that AMG did not modify vasodilatation to ACh. 4. These data suggest that upregulation of gene transcription of the ET system mediates depressed vasorelaxation, NO bioavailability and changes in iNOS and ET(A) receptors that reflect early retinopathy in diabetic rats. Total triterpene acid, in terms of pharmacological properties resembling the endothelin receptor antagonist CPU0213, is effective in normalizing expression of the ET system and iNOS in early diabetic retinopathy and vasculaopathy.

Topics: Animals; Aorta, Thoracic; Aspartic Acid Endopeptidases; Cornus; Diabetes Mellitus, Experimental; Diabetic Retinopathy; Endothelin A Receptor Antagonists; Endothelin-1; Endothelin-Converting Enzymes; Guanidines; Male; Metalloendopeptidases; Nitric Oxide Synthase Type II; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Endothelin A; Retina; RNA, Messenger; Thoracic Arteries; Triterpenes; Up-Regulation; Vasodilation

To investigate the levels of plasma endothelin (ET)-1 and vascular endothelial growth factor (VEGT) in type 2 diabetes mellitus patients, evaluate their clinical application values in different clinical types of diabetic retinopathy (DR), and to investigate the correlation between the levels of plasma ET-1, serum VEGF and the types of DR.. Peripheral blood samples were collected from 240 patients with type 2 diabetes mellitus, 96 with retinopathy and 144 without retinopathy as controls, diagnosed by ophthalmoscopy and fundus fluorescein angiography. The level of plasma ET-1 was detected with radioimmunoassay, and the serum VEGF was measured by ELISA. Pearson correlation analysis and receiver operating characteristic (ROC) curve analysis were conducted.. The expression plasma ET-1 levels of the DM patients with mild non-proliferative retinopathy (NPDR), moderate and severe NPDR and proliferative diabetic retinopathy (PDR), were (178+/-24), (197+/-51), and (231+/-77) ng/L respectively (F=12.186, P<0.01).; and all significantly higher than that of those without DR [(155+/-26) ng/L, all P<0.01]. There was no difference in the expression of VEGF in different courses of DR [(31+/-10), (31+/-8), and (32+/-10) ng/L respectively, F=1.329, P>0.05]. The plasma ET-1 was positively correlated with the type of DR (r=0.504, P<0.01) and the courses of DR (r=0.291, P<0.01). There was a negative correlation between the VEGF level and the type of DR (r=-0.252, P>0.01). ROC curve analysis showed that the plasma ET-1 curve lied in the top left corner, and the curve of VEGF lied under the chance diagonal in all the three different types of DR. The under-curve area of plasma ET-1 were 0.742 at the level of mild retinopathy, 0.723 at the level of moderate and severe NPDR, and 0.857 at the level of PDR. The under-curve areas of VEGF were 0.296, 0.297, and 0.293 respectively. The cut-off points in different types of DR were 173, 197, and 180 ng/L respectively.. Plasma ET-1 is superior to VEGF in diagnosing different types of DR. The expression of VEGF in the blood may not accord with that in the retina. The pathogenetic condition of DR in type 2 diabetes mellitus is aggravated with the course of DR.

Topics: Adult; Aged; Diabetes Mellitus, Type 2; Diabetic Retinopathy; Endothelin-1; Enzyme-Linked Immunosorbent Assay; Female; Humans; Male; Middle Aged; Predictive Value of Tests; Prospective Studies; Radioimmunoassay; ROC Curve; Vascular Endothelial Growth Factor A

Altered activity of retinal endothelin-1 (ET-1) and nitric oxide may play a causal role in the hemodynamic and histopathological changes of diabetic retinopathy. This study evaluated the therapeutic potential of long-term selective blockade of the ET-1(A) receptor (ETRA) to prevent the development of retinopathy in a genetic mouse model of nonobese type 1 diabetes (NOD). Mice with NOD that received subcutaneous implantation of insulin pellets and wild-type control mice were treated for 4 months with the selective ETRA antagonist LU208075 (30 mg/kg/day) via drinking water. At the end of the study, blood glucose levels were evaluated, and animals were anesthetized and perfused intracardially with FITC-labeled dextran. Retinas were removed and either fixed in formalin for confocal microscope evaluation of retinal vascular filling or transferred to RNALater for quantitative reverse transcriptase-polymerase chain reaction to evaluate expression of NOS-3, NOS-1, ET-1, ETRA, ETRB, and the angiogenic factor adrenomedullin. Compared with wild-type controls, expression of ET-1, ETRA, ETRB, and adrenomedullin in mice with NOD were markedly upregulated in the retinas of nontreated mice (cycle time values relative to GAPDH [deltaCt], 14.8 vs. 13.7, 18.57 vs. 17.5, 10.76 vs. 9.9, and 11.7 vs. 9.1, respectively). Mean integral fluorescence intensity (MIFI) of retinal vascular filling was reduced from normal values of 24 to 12.5 in nontreated animals. LU208075 treatment normalized the upregulated expression of ET-1 and adrenomedullin, as well as the deficit in MIFI, but did not affect the increased ETRA and ETRB expression or the elevated plasma glucose levels found in nontreated animals. NOS isoform expression was essentially unchanged. ETRA antagonists may provide a novel therapeutic strategy to slow or prevent progression of retinal microvascular damage and proliferation in patients for whom there is clear evidence of activation of the ET-1 system.

Topics: Adrenomedullin; Animals; Blood Glucose; Diabetic Retinopathy; Endothelin A Receptor Antagonists; Endothelin-1; Female; Hypoglycemic Agents; Injections, Subcutaneous; Insulin; Mice; Mice, Inbred NOD; Peptides; Phenylpropionates; Pyridazines; Time Factors; Vasodilator Agents

To test the hypothesis that bosentan (a dual ET(A)/ET(B) receptor antagonist) corrects a subnormal retinal oxygenation response in the STZ-induced diabetic rat.. In benchtop experiments, ET-1 was acutely injected into the vitreous of control and 5- to 7-day bosentan-treated nondiabetic rats. Major retinal vessel diameters were analyzed from ADPase-stained flatmounts. Retinal oxygenation (DeltaPo(2)), an established early surrogate marker of drug treatment efficacy, was measured by MRI during a 2-minute carbogen inhalation challenge in four groups: control rats (n = 7), control rats treated with bosentan (n = 7), 3-month diabetic rats (n = 9), and 3-month diabetic rats treated with bosentan (n = 5). Effect of baseline differences was studied in control rats breathing either room air (n = 5) or 12% oxygen breathing (n = 5) before a 2-minute carbogen provocation.. ET-1 produced a significant (P < 0.05) reduction in retinal arterial diameter that was suppressed (P > 0.05) in rats fed bosentan chow admix. For all groups, no MRI baseline signal intensity differences were found (P > 0.05). Also, comparisons between baseline room air and 12% conditions and control rats fed normal chow or a bosentan admix both produced similar (P > 0.05) panretinal DeltaPo(2). In treated and untreated diabetes groups, inferior hemiretinal DeltaPo(2) remained normal (P > 0.05), but superior hemiretinal DeltaPo(2) was subnormal (P < 0.05).. Because subnormal retinal DeltaPo(2) after drug treatment is a biomarker of subsequent vascular histopathology, the present data raise the possibility that retinal ET-1 does not play a key role in the pathogenesis of diabetic retinopathy.

Topics: Animals; Apyrase; Bosentan; Diabetes Mellitus, Experimental; Diabetic Retinopathy; Endothelin A Receptor Antagonists; Endothelin B Receptor Antagonists; Endothelin-1; Magnetic Resonance Imaging; Oxygen; Oxygen Consumption; Partial Pressure; Rats; Retina; Retinal Vessels; Sulfonamides

Preferential expression of oncofetal extra domain-B fibronectin (EDB(+) FN), a proposed angiogenic marker, has been shown in proliferative diabetic retinopathy. High levels of glucose also increase EDB(+) FN expression in endothelial cells (ECs) via transforming growth factor-beta1 (TGF-beta1) and endothelin-1 (ET-1). The present study was aimed at elucidating the role of serum- and glucocorticoid-regulated kinase (SGK-1) in glucose-induced EDB(+) FN expression. Using human macro- and microvascular ECs, we show that high levels of glucose, TGF-beta1, and ET-1 increase the EDB(+) FN expression via SGK-1 alteration at the mRNA, protein, and activity levels. Inhibition of TGF-beta1 and ET-1 prevented glucose-induced SGK-1 activation and the EDB(+) FN expression. Furthermore, using siRNA-mediated SGK-1 gene silencing, we show that glucose-induced EDB(+) FN expression can be completely prevented. These findings provide first evidence of glucose-induced SGK-1 activation in altered EDB(+) FN expression and provide novel avenues for therapeutic modalities.

Topics: Blotting, Western; Cells, Cultured; Diabetic Retinopathy; Endothelin-1; Endothelium, Vascular; Enzyme Activation; Fibronectins; Gene Silencing; Glucose; Humans; Immediate-Early Proteins; Immunoblotting; Microcirculation; Microscopy, Fluorescence; Nuclear Proteins; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Time Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Umbilical Veins

Activation of protein kinase C (PKC) has been implicated in the pathogenesis of diabetic retinopathy. The purpose of this study was to investigate the role of PKC on the alteration of retinal endothelin-1 in 2-week streptozotocin (STZ)-induced diabetic rats. The measurement of retinal PKC activities from membranous and cytosolic fractions was conducted by ELISA. Retinal tissues were analysed for the expression of endothelin-1 (ET-1), endothelin-3 (ET-3), endothelin-A (ET-A), and endothelin-B (ET-B) mRNA by means of semi-quantitative RT-PCR. Retinal vasculature isolated by trypsin digest technique was immunostained for ET-1. We followed the alteration of retinal ET-1 after intravitreal injection of a general PKC inhibitor, GF109203X, in 2-week diabetic rats. Retinal PKC specific activities were significantly increased by 37% (P=0.027) in the membranous fraction in diabetic rats compared with normal rats, whereas PKC specific activities in the cytosolic fraction were unchanged. The retina from the diabetic rats showed increased ET-1 mRNA expression after 2 weeks, while no changes were found for ET-3, ET-A and ET-B. ET-1 immunoreactivity was also increased in the retinal vasculature of diabetic rats. Retinal ET-1 expression was decreased after intravitreal injection of GF109203X (10(-5), 10(-6), 10(-7) M) in a dose-dependent manner. The results from this study showed that the enhanced ET-1 expression associated with the activation of PKC has occurred in early diabetes, and PKC inhibitor could reverse the up regulation of ET-1.

Topics: Animals; Cell Membrane; Cytosol; Diabetes Mellitus, Experimental; Diabetic Retinopathy; Dose-Response Relationship, Drug; Endothelin-1; Endothelins; Enzyme Inhibitors; Gene Expression Regulation; Indoles; Male; Maleimides; Protein Kinase C; Rats; Rats, Sprague-Dawley; Retina; Retinal Vessels; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Imbalance between extracellular matrix protein synthesis and degradation is a key feature of diabetic retinopathy. Fibronectin, a predominant constituent of the extracellular matrix, has been shown to undergo alternative splicing to produce embryonic isoforms in various pathologic conditions, such as fibrotic diseases and tumorigenesis. Two such isoforms, oncofetal fibronectin variants that are characterized by the inclusion of the oncofetal domains A and B, were the focus of the present study.. The expression of oncofetal fibronectin variants was determined in human vitreous samples obtained from patients undergoing vitrectomy for proliferative diabetic retinopathy and nondiabetes-associated ocular conditions such as macular hole. In addition, an animal model of chronic diabetes and cultured endothelial cells was used to elucidate the mechanistic basis for this aberrant expression of oncofetal fibronectin.. Expression of fibronectin containing the oncofetal domain B was upregulated in the vitreous of patients with diabetic retinopathy.. Use of a well-established animal model of chronic diabetic complications and cultured endothelial cells showed that diabetes-induced upregulation of oncofetal fibronectin is, in part, dependent on hyperglycemia-induced transforming growth factor-beta1 and endothelin-1. Furthermore, the data suggest that oncofetal fibronectin is involved in endothelial cell proliferation.

Topics: Aged; Animals; Blotting, Western; Bosentan; Cell Division; Cells, Cultured; Diabetes Mellitus, Experimental; Diabetic Retinopathy; Endothelin-1; Endothelium, Vascular; Female; Fibronectins; Gene Silencing; Humans; Male; Middle Aged; Rats; Rats, Sprague-Dawley; Retinal Perforations; Reverse Transcriptase Polymerase Chain Reaction; Sulfonamides; Transforming Growth Factor beta; Transforming Growth Factor beta1; Up-Regulation; Vitrectomy; Vitreous Body

Understanding the causes of diabetic vascular complications has become an increasingly important issue because of the rapidly rising prevalence of diabetes. Recently discovered vasoconstrictors and angiogenesis regulators, such as endothelin (ET) and vascular endothelial growth factor (VEGF), have been intensely studied for possible pathogenic roles in diabetic vascular complications. The present study was undertaken to clarify the effect of glycemic control on serum VEGF and plasma ET-1 concentrations in diabetic patients, and to identify other factors that may cause fluctuations of these substances. Plasma VEGF and ET-1 concentrations of 45 hospitalized diabetic patients and 54 control subjects were measured by enzyme immunoassay (EIA) and radioimmunoassay (RIA), respectively. Plasma VEGF was elevated in poorly controlled diabetic patients compared with healthy subjects and plasma VEGF concentrations declined after hospitalized treatment with either insulin or oral hypoglycemic agents in combination with diet. There was a significant correlation between plasma VEGF concentration and both fasting plasma glucose (FPG) and hemoglobin A(1c) (HbA(1c)). Plasma ET-1 in poorly controlled diabetic patients was higher than in healthy controls, but improved glycemic control did not affect plasma ET-1 concentrations. Thus, poor glycemic control causes increased levels of plasma VEGF, which may result in hypertension and vascular complications in diabetes. Short-term treatment resulting in good glycemic control can improve levels of VEGF and may provide beneficial effects on diabetic vascular complications.

Topics: Adult; Aged; Blood Glucose; Body Mass Index; Case-Control Studies; Diabetes Complications; Diabetes Mellitus; Diabetic Neuropathies; Diabetic Retinopathy; Endothelin-1; Fasting; Female; Glycated Hemoglobin; Humans; Hypoglycemic Agents; Male; Middle Aged; Proteinuria; Regression Analysis; Vascular Endothelial Growth Factor A

Increased vascular permeability and blood flow alterations are characteristic features of diabetic retinal microangiopathy. The present study investigated vascular endothelial growth factor (VEGF) and its interactions with endothelin (ET) 1 and 3, endothelial, and inducible nitric oxide synthase (eNOS, iNOS) in mediating diabetes induced retinal vascular dysfunction. Male Sprague Dawley rats with streptozotocin (STZ) induced diabetes, with or without VEGF receptor signal inhibitor SU5416 treatment (high or low dose) were investigated after 4 weeks of follow-up. Colour Doppler ultrasound of the ophthalmic/central retinal artery, retinal tissue analysis with competitive RT-PCR and microvascular permeability were studied. Diabetes caused increased microvascular permeability along with increased VEGF mRNA expression. Increased vascular permeability was prevented by SU5416 treatment. Diabetic animals showed higher resistivity index (RI), indicative of vasoconstriction with increased ET-1 and ET-3 mRNA expression, whereas eNOS and iNOS mRNA expressions were un-affected. SU5416 treatment corrected increased RI via increased iNOS in spite of increased ET-1, ET-3 and VEGF mRNA expression. Cell culture (HUVEC) studies indicate that in part, an SU5416 induced iNOS upregulation may be mediated though a MAP kinase signalling pathway. The present data suggest VEGF is important in mediating both vasoconstriction and permeability in the retina in early diabetes.

Topics: Animals; Capillary Permeability; Diabetes Mellitus, Experimental; Diabetic Retinopathy; Endothelin-1; Endothelin-3; Indoles; Male; MAP Kinase Signaling System; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Pyrroles; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Vascular Endothelial Growth Factor A

Increased expression of endothelin-1 (ET-1) is associated with diabetic retinopathy and vasculopathy, although the molecular explanation has not been defined. The effects of high glucose and protein kinase C (PKC) activation on platelet-derived growth factor (PDGF)-BB and of ET-1 expression in the retina of streptozotocin (STZ)-induced diabetic rats and bovine retinal pericytes (BRPC) were examined. In 4-week diabetic rats, PDGF-B and prepro-ET-1 (ppET-1) mRNA levels increased significantly by 2.8- and 1.9-fold, respectively, as quantified by RT-PCR. Treatment with PKC-beta isoform-specific inhibitor (LY333531) or insulin normalized retinal ET-1 and PDGF-B expression. In BRPC, high glucose levels increased ppET-1 and PDGF-B mRNA expression by 1.7- and 1.9-fold, respectively. The addition of PDGF-BB but not PDGF-AA increased expression of ppET-1 and vascular endothelial growth factor mRNA by 1.6- and 2.1-fold, respectively, with both inhibited by AG1296, a selective PDGF receptor kinase inhibitor. A general PKC inhibitor, GF109203X, suppressed PDGF-BB's induction of ET-1 mRNA. Thus, increased ET-1 expression in diabetic retina could be due to increased expression of PDGF-BB, mediated via PDGF-beta receptors in part by PKC activation. The novel demonstration of elevated expression of PDGF-B and its induction by PKC activation identifies a potential new molecular step in the pathogenesis of diabetic retinopathy.

Topics: Animals; Becaplermin; Capillaries; Cells, Cultured; Diabetes Mellitus, Experimental; Diabetic Retinopathy; Endothelial Growth Factors; Endothelin Receptor Antagonists; Endothelin-1; Enzyme Activation; Enzyme Inhibitors; Gene Expression; Indoles; Insulin; Intercellular Signaling Peptides and Proteins; Lymphokines; Male; Maleimides; Mitogen-Activated Protein Kinase Kinases; Platelet-Derived Growth Factor; Protein Kinase C; Protein Kinase C beta; Proto-Oncogene Proteins c-sis; Rats; Rats, Sprague-Dawley; Receptor, Endothelin A; Retina; Retinal Vessels; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Both insulin and IGF-1 have been implicated in control of retinal endothelial cell growth, neovascularization, and diabetic retinopathy. To precisely define the role of insulin and IGF-1 signaling in endothelium in these processes, we have used the oxygen-induced retinopathy model to study mice with a vascular endothelial cell-specific knockout of the insulin receptor (VENIRKO) or IGF-1 receptor (VENIFARKO). Following relative hypoxia, VENIRKO mice show a 57% decrease in retinal neovascularization as compared with controls. This is associated with a blunted rise in VEGF, eNOS, and endothelin-1. By contrast, VENIFARKO mice show only a 34% reduction in neovascularization and a very modest reduction in mediator generation. These data indicate that both insulin and IGF-1 signaling in endothelium play a role in retinal neovascularization through the expression of vascular mediators, with the effect of insulin being most important in this process.

Topics: Animals; Diabetic Retinopathy; Disease Models, Animal; Endothelial Growth Factors; Endothelin-1; Endothelium, Vascular; Humans; Hypoxia; Insulin; Intercellular Signaling Peptides and Proteins; Lymphokines; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Mice, Knockout; Neovascularization, Pathologic; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Receptor, IGF Type 1; Receptor, Insulin; Retinal Vessels; Signal Transduction; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Heme oxygenase (HO) isoforms, HO-1, and HO-2, are responsible for heme breakdown to iron and carbon monoxide (CO). HO may respond to oxidative stress and may modulate the expression of vasoactive factors like nitric oxide (NO). Since diabetes induced oxidative stress may change HO, the present study examined whether diabetes is associated with HO alterations, its relationship with NO, endothelin-1(ET-1) and the functional significance.. Male SD rats with Streptozotocin induced diabetes were investigated after six-weeks. Poorly controlled diabetic animals were randomized to one of three treatment groups (n = 6 each group); a) untreated, b) HO-1 inhibitor SnPP-IX (50 micromol/kgIP/day), c) NO donor molsidomine (120 mg/L PO/day) and were compared with age and sex matched non diabetic control animals with or without SnPP-IX treatment. Color Doppler ultrasound analysis was used to determine retinal resistivity index (RI). mRNA for HO-1, HO-2, ET-1, eNOS and iNOS were analyzed with competitive RT-PCR. HO distribution in the retina was investigated by immunocytochemistry.. Diabetic animals expressed lower body weight, higher blood glucose and increased glycated hemoglobin levels. HO-1 and HO-2 immuno-reactivity were identified in the retina. Diabetes induced increased RI was associated with up-regulation of both ET-1 and HO-1 mRNA expression but not eNOS or iNOS mRNA. Both SnPP-IX and molsidomine treatments prevented a diabetes increase of RI, in spite of increased ET-1 expression and were associated with increased iNOS mRNA.. The present data suggests that the HO system is up-regulated in short term diabetes leading to HO and NO interactions which may modulate vascular function in the retina.

Topics: Animals; Blood Glucose; Body Weight; Diabetes Mellitus, Experimental; Diabetic Retinopathy; Endothelin-1; Enzyme Inhibitors; Glycated Hemoglobin; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Male; Metalloporphyrins; Molsidomine; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Protoporphyrins; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Up-Regulation

It has been reported that improvement of metabolic control by intensified insulin therapy in patients with Type I (insulin-dependent) diabetes mellitus is associated with alterations in ocular blood flow. We hypothesized that these changes in ocular blood flow could be associated with alterations of plasma insulin, glucose or endothelin concentration.. In 16 patients with Type I diabetes ocular haemodynamic parameters were assessed daily during the first 5 days of institution of intensified insulin therapy and plasma concentrations of glucose, insulin, and endothelin-1 plasma were measured. Retinal white blood cell flux was estimated with the blue field entoptic technique. Pulsatile choroidal blood flow was assessed by laser interferometric measurement of fundus pulsation amplitude.. Retinal white blood cell flux ( p=0.0015) and ocular fundus pulsation amplitude ( p<0.001) increased during institution of strict metabolic control. Changes in ocular haemodynamic variables were inversely correlated with concentrations of plasma ET-1, but not with that of insulin or glucose.. These data indicate that institution of improved metabolic status is paralleled by rapid changes in the production of ET-1, which could in turn affect ocular perfusion.

Topics: Adult; Albuminuria; Blood Glucose; Blood Pressure; Cholesterol, HDL; Diabetes Mellitus, Type 1; Diabetic Retinopathy; Dose-Response Relationship, Drug; Endothelin-1; Female; Hemodynamics; Humans; Hypoglycemic Agents; Insulin; Male; Middle Aged; Pulse; Retinal Vessels; Triglycerides

We have investigated the localisation of endothelin-1 (ET-1) mRNA and ET-1-like immunoreactivity in retina and anterior portion of optic nerve from human and porcine eyes. In situ hybridisation method revealed expression of ET-1 mRNA mainly in the innermost layers of the retinas, in the retinal pigment epithelium cells as well as in the astrocytes of the optic nerve. Immunohistochemical studies showed that ET-1-like immunoreactivity appeared in the same regions where ET-1 mRNA was expressed as well as in the inner nuclear layer and in the inner segments of photoreceptors. In the nerve fibre and ganglion cell layers, astrocytes expressed both glial fibrillary acidic protein and ET-1 proteins suggesting that these cells may secrete ET-1. Expression of ETA and ETB receptors in human retina were demonstrated by reverse transcription-polymerase chain reaction. Our results demonstrated expression of ET-1 in glial, neural and vascular components of retina and optic nerve from human and porcine eyes.

Topics: Aged; Animals; Astrocytes; Diabetic Retinopathy; DNA, Complementary; Endothelin-1; Female; Glial Fibrillary Acidic Protein; Humans; Immunohistochemistry; Male; Middle Aged; Muscle, Smooth, Vascular; Neurons; Optic Nerve; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin; Retina; Retinal Artery; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Swine; von Willebrand Factor

Patients with type 1 diabetes, aged 18 to 42 years, were compared to those aged 11 to 22 years. Activities of endothelial vasoactive factors and endothelial and leukocyte adhesion molecules were studied at different stages of development diabetes complications: nephropathy and retinopathy. The findings reveal role of the vasoactive factors in microangiopathy course.

Topics: Adolescent; Adult; Biomarkers; Child; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Diabetic Retinopathy; E-Selectin; Endothelin-1; Endothelium, Vascular; Epoprostenol; Humans; Intercellular Adhesion Molecule-1; Microcirculation; Nitric Oxide; Thromboxane A2

Overproduction of endothelin-1 (ET-1) and nitric oxide (NO) in the retina is demonstrated in experimental diabetic animals. To clarify the possible involvement of ET-1 and NO in the pathogenesis of diabetic retinopathy, the authors examined the vitreous levels of these principal endothelium-derived vasoactive substances in patients with proliferative diabetic retinopathy (PDR).. Vitreous fluid was taken from patients with PDR (ET-1, n = 12; NO, n = 12) and from patients with macular holes as controls (ET-1, n = 10; NO, n = 10) at vitreous surgery. Endothelin-1 and NO metabolites were measured by radioimmunoassay and high-performance liquid chromatography based on the Griess method, respectively.. Endothelin-1 levels (mean +/- SE) were 21.5 +/- 1.7 pg/mL in the vitreous of patients with PDR and 16.7 +/- 0.7 pg/mL in the vitreous of patients with macular hole. There was a significant difference between patients with PDR and controls (P = 0.009, Mann-Whitney). Nitrate (NO3) was 49.8 +/- 5.0 micromol/L in patients with PDR and 24.2 +/- 2.8 micromol/L in patients with macula hole; it was also significantly elevated in patients with PDR (P = 0.004, Mann-Whitney), whereas nitrite (NO2) was not detected in this study.. These results indicate that ET-1 and NO may be related in the pathogenesis of PDR.

Topics: Adult; Aged; Aged, 80 and over; Chromatography, High Pressure Liquid; Diabetic Retinopathy; Endothelin-1; Female; Humans; Male; Middle Aged; Nitrates; Nitric Oxide; Nitrites; Radioimmunoassay; Retinal Perforations; Vitreous Body

The endothelin system (ET system) has been implicated in the retinal blood flow abnormalities that precede the onset of diabetic retinopathy. This study was undertaken to assess whether the density and localisation of both the immunoreactive endothelin-1 and endothelin receptors in rat retina change in the early stages of diabetes and the insulin treatment would affect those changes.. Untreated streptozotocin-diabetic, insulin-treated streptozotocin-diabetic and age-matched control rats were killed 15, 45 and 90 days after the induction of diabetes. Binding assays were used to determine the density and proportion of endothelin receptors in neural retinal membranes. Localisation of endothelin receptors and immunoreactive endothelin-1 were analysed by microautoradiography and immunohistochemistry, respectively.. Fifteen days after the induction of diabetes, the neural retinal membranes of untreated streptozotocin-diabetic rats showed a statistically significant decrease in the density of both endothelin receptor subtypes when compared with age-matched control rats. At 90 days, however, the density of endothelin receptors type B was statistically significantly higher than that of control rats, and the innermost layers of the diabetic retina also showed an increase of both endothelin receptor type B receptor and immunoreactive endothelin-1 signal. Insulin treatment during 90 days up-regulated endothelin receptor type A in neural retinal membranes and in the innermost layers of the retina when compared with control retinas.. These results show that the endothelin system is altered in both vascular and neuronal components of the retina in early diabetic retinopathy. The up-regulation of endothelin receptor type A induced by insulin treatment suggests that insulin might be involved in retinal microangiopathy.

Topics: Animals; Autoradiography; Diabetes Mellitus, Experimental; Diabetic Retinopathy; Endothelin-1; Immunohistochemistry; Insulin; Iodine Radioisotopes; Male; Neurons; Rats; Rats, Wistar; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin; Retina; Time Factors

Endothelin-1, and constitutive endothelial nitric oxide synthase, have been implicated in the pathogenesis of diabetic retinopathy. We therefore screened polymorphisms within the genes encoding these two vasoactive agents in a sample of individuals with 15 years of diabetes and no retinopathy (ETDRS level 10 or better) and those with severe retinopathy (ETDRS level 50 or worse).. PCR primers for highly polymorphic sites within the EDN1 and NOS3 genes were used to genotype individuals with type 1 or type 2 diabetes with severe or no retinopathy. Allele frequencies were compared between groups using chi-squared analysis and adjusting for multiple comparisons.. No significant differences were observed in allele frequencies for these two markers between the patients who had retinopathy and the patients who did not.. Polymorphic variability in the EDN1 and NOS3 genes does not appear to have a major impact on determining susceptibility or resistance to diabetic retinopathy.

Topics: Adult; Alleles; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diabetic Retinopathy; Dinucleotide Repeats; Endothelin-1; Genetic Predisposition to Disease; Humans; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Polymerase Chain Reaction; Polymorphism, Genetic

Raised circulating levels of the potent vasoconstrictor endothelin-1 (ET-1) have been demonstrated in diabetes and pregnancy-induced hypertension. Pregnant women with diabetic retinopathy are known to be at a higher risk of developing pregnancy-induced hypertension than those without retinopathy. To examine the association between ET-1, pregnancy, diabetes and diabetic retinopathy, circulating ET-1 levels were measured in each trimester in a cohort of women with and without diabetes during pregnancy.. A cohort of women with diabetes (n = 13) and normal controls (n = 8) were followed throughout pregnancy. Detailed clinical and fundoscopic examinations were carried out according to ETDRS protocols. Plasma ET-1 levels were measured in each trimester using a sensitive radioimmunoassay. Those with diabetes were further divided into those with retinopathy (n = 7) and those without (n = 6).. Plasma levels of ET-1 increased progressively during normal pregnancy and peaked in the third trimester. Women with diabetes had significantly higher levels of plasma ET-1 (14.0 vs 4.6 pg/ml in the first trimester, 14.0 vs 4.8 pg/ml in the second trimester and 15.8 vs 7.2 pg/ml in the third trimester) compared with those without diabetes. These were no significant differences in plasma ET-1 levels between women with diabetes who had pre-existing diabetic retinopathy and those without.. This study has shown that ET-1 levels rise during normal pregnancy, and are higher in women with diabetes, which may reflect pre-existing endothelial damage. Although no association could be demonstrated between diabetic retinopathy and serum ET-1 levels, this may reflect the small sample size in this study.

Topics: Adult; Biomarkers; Cohort Studies; Diabetes Mellitus, Type 1; Diabetic Retinopathy; Disease Progression; Endothelin-1; Female; Humans; Pregnancy; Pregnancy in Diabetics; Radioimmunoassay

Vasoactive factors like endothelins, by virtue of the microvascular regulation as well as by other effects, possibly play important parts in the pathogenesis of diabetic retinal microangiopathy. We investigated retinal vascular dysfunction in streptozotocin-induced diabetes and its relation with endothelins in short- and long-term diabetes.. Diabetic rats with or without an endothelin receptor antagonist (bosentan) treatment were investigated after 1 month and 6 months of follow-up. Retinal blood flow was measured and compared with age- and sex-matched non-diabetic control animals. Retinal tissues were analysed for endothelin-1, endothelin-3, endothelin A and endothelin B mRNA. Distribution of endothelin-1 and endothelin-3 was investigated by immunocytochemistry and that for endothelin receptors by ligand binding and autoradiography.. Diabetic animals showed hyperglycaemia, glycosuria, elevated glycated haemoglobin values and reduced body weight gain. Retinal blood flow showed an increased resistivity index, an indicator of vasoconstriction, after 1 month of diabetes which was prevented by treatment with bosentan. This functional change in diabetes was eliminated after 6 months of follow-up. The retina from the diabetic animals showed increased mRNA expression for endothelin-1, endothelin-3 and endothelin A after one month. In addition, endothelin B mRNA expression was increased after 6 months. Furthermore, endothelin-1 and endothelin-3 immunoreactivity and endothelin receptor concentrations were increased in the retina of diabetic rats.. The results from this study indicate that the endothelin system is of importance in mediating retinal changes in diabetes although mechanisms of the endothelin system alteration as well as their effects might vary depending on the duration of diabetes. [Diabetologia (1999) 42: 1228-1234]

Topics: Animals; Antihypertensive Agents; Autoradiography; Bosentan; Diabetes Mellitus, Experimental; Diabetic Retinopathy; Endothelin Receptor Antagonists; Endothelin-1; Endothelin-3; Immunohistochemistry; Male; Rats; Rats, Sprague-Dawley; Receptor, Endothelin A; Receptor, Endothelin B; Retina; Retinal Vessels; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sulfonamides; Ultrasonography, Doppler, Color

Topics: Diabetic Retinopathy; Endothelin-1; Humans

Endothelins (ETs) belong to a family of vasoactive peptides implicated in several disorders of the microvasculature. In the present study, we investigated ET-1 and ET-3 peptide mRNAs and ETA, ETB receptor mRNAs in the retina of diabetic BB/W rats and age-matched, non-diabetic control animals, following six months of diabetes.. Total mRNA was extracted from each retina and was subjected to reverse transcriptase polymerase chain reaction for ET-1, ET-3, ETA and ETB. Simultaneously, beta-globin was amplified and used as a housekeeping gene. The products were analyzed on agarose gels and the specificity of the amplification was established by hybridization with amplification-specific biotinylated oligoprobes. For quantification, the products from the linear phase of amplification were subjected to serial dilution slot-blot hybridization and densitometry.. ETs and their receptor mRNA expressions were present in the retina. Retinas from the diabetic animals showed significant increases in ET-1, ET-3 ET(A), ET(B) mRNA expressions compared to those from control rats.. These findings indicate that retinal ET-1, ET-3, ET(A) and ET(B) mRNA expression in increased in the chronically diabetic BB/W rat. Augmented gene expression of ETs and their receptors potentially may be of importance in the pathogenesis of retinal microangiopathy in diabetes.

Topics: Animals; Blotting, Southern; Chronic Disease; Diabetes Mellitus, Type 1; Diabetic Retinopathy; DNA Primers; DNA, Antisense; Endothelin-1; Endothelin-3; Endothelins; Gene Expression; Male; Polymerase Chain Reaction; Rats; Rats, Inbred BB; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin; Retina; RNA; RNA, Messenger

Endothelin-1 (ET-1), a novel 21-amino acid vasoconstrictive peptide secreted by endothelial cells, has been thought to play a role in various forms of vascular disease. Diabetes mellitus is well known for its association with microvascular damage. To investigate whether ET-1 levels may be related to microangiopathy in diabetes mellitus, plasma ET-1 levels were measured in two groups of diabetic patients: A) 47 patients with non-insulin dependent diabetes mellitus (NIDDM) and retinopathy (28 M, 19 F; mean age 60.7+/-8.5 yrs) but without nephropathy (microalbuminuria < 30 mg/day) and hypertension (SBP < 140, DBP < 90 mmHg); group A was divided in three subgroups based on the severity of retinopathy: a) 16 with background retinopathy; b) 21 with pre-proliferative retinopathy; c) 10 with proliferative retinopathy. B) 8 patients with insulin-dependent diabetes mellitus (IDDM) recently diagnosed (6 M, 2 F; 16.4+/-3.8 yrs) without complications. C) 28 healthy subjects (HS) (16 M, 12 F; 47.8+/-11.8 yrs) as controls. In the NIDDM group the ET-1 concentration was significantly higher (17.3+/-2.4 pg/ml) than both in the HS (8+/-4.7 pg/ml) and IDDM patients (10.2+/-3.7 pg/ml) (p < 0.0001). In the subgroups with retinopathy the ET-1 levels were a) 15.1+/-4.3 pg/ml; b) 22.2+/-6.8 pg/ml and c) 16.6+/-5.1 pg/ml. These values were significantly elevated as compared to HS (p<0.001; p < 0.0001; p < 0.002, respectively), being the highest levels of ET-1 observed in the NIDDM patients with pre-proliferative retinopathy. In conclusion our study revealed that the ET-1 concentrations are elevated in NIDDM patients with retinopathy especially in those patients with pre-proliferative retinopathy.

Topics: Aged; Diabetes Mellitus, Type 2; Diabetic Retinopathy; Endothelin-1; Female; Humans; Male; Middle Aged

Increased levels of endothelin (ET-1), a potent endothelium-derived vasoconstrictive peptide, have been found in plasma from non-insulin dependent diabetic (NIDDM) patients, suggesting that ET-1 might represent a new marker of diabetes-related vascular damage. To elucidate this topic, circulating ET-1 levels were evaluated in 16 NIDDM patients in good metabolic control without either cardiovascular risk factors (obesity, hypertension, smoking, hyperdislipidaemia, etc.) or diabetes-related damage of other districts and in 12 healthy subjects. Retinopathy was assessed by ophthalmological evaluation and its severity determined by Klein criteria. Resulting data showed higher levels of plasma ET-1 in NIDDM patients than in control subjects (0.80 +/- 0.13 vs 0.60 +/- 0.12 pg/mL, p < 0.001). Plasma ET-1 levels were directly correlated with retinopathy degrees in NIDDM patients affected by retinopathy (n = 10; r = 0.368; p = 0.02), and were significantly higher in these latter (n = 10) than in those without retinopathy (n = 6) (0.89 +/- 0.13 vs 0.71 +/- 0.19 pg/mL, p < 0.05). The increased levels of ET-1 could contribute to retinopathy development or, more probably, represent a marker of this diabetes-related complication.

Topics: Adult; Biomarkers; Cardiovascular Diseases; Diabetes Mellitus, Type 2; Diabetic Retinopathy; Endothelin-1; Humans; Male; Middle Aged; Reference Values; Risk Factors

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.. 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.. 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.. 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.

Topics: Animals; Chromatography, High Pressure Liquid; Diabetes Mellitus, Experimental; Diabetic Retinopathy; Endothelin-1; Endothelin-3; Insulin; Intestine, Small; Kidney; Male; Radioimmunoassay; Rats; Rats, Wistar; Retina; Retinal Vessels

To investigate the role of endogenous endothelin-1 (ET-1) expression and its interaction with the ETA receptor in the physiologic regulation of vascular tone as well as in the development of abnormal retinal hemodynamics in diabetes.. Retinal blood flow, using digitized video fluorescein angiography recordings, was quantitated after intravitreous injections of ET-1; BQ-123, an ETA receptor antagonist; and phosporamindon, an endothelin converting enzyme inhibitor in the eyes of diabetic and nondiabetic rats. A total of 154 rats were used for these experiments. Message levels of preproendothelin-1 (preproET-1) were measured from the retina of diabetic and nondiabetic rats using competitive polymerase chain reaction (PCR) techniques.. Retinal blood flow was reduced (33%, P < 0.001) in diabetic rats compared to nondiabetic rats. BQ-123, an ETA receptor antagonist, but not saralasin, an angiotensin receptor antagonist, increased retinal blood flow in a dose-dependent manner in diabetic (EC50 of 8 x 10(-7) M) and in nondiabetic rats (EC50 of 8 x 10(-8) M). Besides being resistant to BQ-123, the maximal response in diabetic animals occurred 20 minutes later than in nondiabetic animals. Decreasing ET-1 levels by inhibiting endothelin-converting enzyme with phosphoramidon normalized retinal blood flow in diabetic rats. In nondiabetic rats, the intravitreous injection of exogenous ET-1 (10(-8) M) resulted in retinal blood flow decreases comparable to those measured in diabetic animals, and the subsequent injection of 10(-4) M BQ-123 produced retinal blood flow changes comparable to those measured in BQ-123 injected diabetic rats. Comparison of preproET-1 messenger RNA expression in the retina, brain and lung of control and diabetic rats using quantitative PCR and Northern blot analysis showed 2.0- and 1.7-fold increases in the retina and the brain, respectively, without changes in the lung.. These data suggest that ET-1 is involved in the regulation of retinal blood flow in normal physiologic outcome, and an increase in the endogenous expression of ET-1 contributes to the reduction of retinal blood flow reported in the early stages of diabetes mellitus.

Topics: Animals; Brain; Diabetes Mellitus, Experimental; Diabetic Retinopathy; DNA Primers; Dose-Response Relationship, Drug; Endothelin Receptor Antagonists; Endothelin-1; Endothelins; Fluorescein Angiography; Glycopeptides; Hemodynamics; Male; Metalloendopeptidases; Peptides, Cyclic; Polymerase Chain Reaction; Protease Inhibitors; Protein Precursors; Rats; Rats, Inbred BN; Rats, Sprague-Dawley; Receptors, Endothelin; Retina; Retinal Vessels; RNA, Messenger