Apelin-13 Is an Early Promoter of Cytoskeleton and Tight Junction in Diabetic Macular Edema via PI-3K/Akt and MAPK/Erk Signaling Pathways.

Diabetic macular edema is major cause of vision loss associated with diabetic retinopathy. Breakdown of blood-retinal barrier, especially inner BRB, is an early event in pathogenesis of DR. Apelin, an endogenous ligand of APJ, mediates angiogenesis and is involved in the development of DR. The present study aimed to investigate effects and mechanism of apelin-13 in vascular permeability during DME. We verified apelin-13 was upregulated in DME patients' vitreous. High glucose incubation led to a progressive increase of apelin-13, APJ, cytoskeleton, and tight junction proteins, including VE-Cadherin, FAK, Src, ZO-1, and occludin. Apelin-13 promoted HRMEC proliferation and migration and phosphorylation of both cytoskeleton and tight junction under both normal and high glucose conditions. Besides, apelin-13 activated PI-3K/Akt and MAPK/Erk signaling pathways, including PLCγ1, p38, Akt, and Erk both in HRMEC and in C57BL/6 mice. Meanwhile, F13A performed opposite effects compared with apelin-13. In in vivo study, apelin-13 was also upregulated in retina of db/db mice. Taken together, apelin-13 increased biologic activity of HRMEC, as well as expression of both cytoskeleton and tight junction in DME via PI-3K/Akt and MAPK/Erk signaling pathways. Apelin-13 as an early promoter of vascular permeability may offer a new perspective strategy in early treatment of DR.

Benchmarking cataract surgery outcomes in an ethnically diverse and diabetic population: final post-operative visual acuity and rates of post-operative cystoid macular oedema.

PurposeTo determine visual acuity and rates of post-operative cystoid macular oedema (CMO) in an ethnically diverse and predominantly diabetic population.MethodsThe study was undertaken over a one year period. Snellen visual acuity (VA) was measured pre and 4-8 weeks post-operatively and optical coherence topography (OCT) was performed at baseline and post-operatively. No eyes received prophylactic non-steroidal anti-inflammatory drugs (NSAIDs) prior to or after surgery.ResultsOut of 262 eyes, 59% were in the Black, Asian and minority ethnic group (BAME), 57% had a history of diabetes mellitus and 34% had pre-existing diabetic retinopathy. 76% of all eyes achieved 6/12 post-operative VA at the first postoperative review and the incidence of post-operative CMO within the study population was 7.6%. In patients with a history of diabetes mellitus the incidence of post-operative CMO was 10.7% compared to 3.5% in those without diabetes. This was found to be clinically significant (P=0.0297).ConclusionWe believe this is the first study to benchmark standards for care in this demographic of patients. A history of diabetes mellitus, irrespective of whether there is diabetic retinopathy or not, is an independent risk factor for the development of post-operative CMO in patients undergoing cataract surgery (P=0.0297). Ethnicity is not an independent risk factor for the development of post-operative CMO.

Matrix Metalloproteinase-1 and Matrix Metalloproteinase-9 in the Aqueous Humor of Diabetic Macular Edema Patients.

PURPOSE: To assess the concentrations of matrix metalloproteinase (MMP)-1 and MMP-9 in the aqueous humor of diabetic macular edema (DME) patients. METHOD: The concentrations of MMP-1 and MMP-9 in the aqueous humors of 15 cataract patients and 25 DME patients were compared. DME patients were analyzed according to the diabetic retinopathy (DR) stage, diabetes mellitus (DM) duration, pan-retinal photocoagulation (PRP) treatment, recurrence within 3 months, HbA1C (glycated hemoglobin) level, and axial length. RESULTS: The concentrations of MMP-1 and MMP-9 of the DME groups were higher than those of the control group (p = 0.005 and p = 0.002, respectively). There was a significant difference in MMP-1 concentration between the mild non-proliferative diabetic retinopathy (NPDR) group and the proliferative diabetic retinopathy (PDR) group (p = 0.012). MMP-1 concentrations were elevated in PRP-treated patients (p = 0.005). There was a significant difference in MMP-9 concentrations between the mild NPDR group and the PDR group (p < 0.001), and between the moderate and severe NPDR group and the PDR group (p < 0.001). The MMP-9 concentrations in PRP treated patients, DM patients with diabetes ≥ 10 years and recurrent DME within 3months were elevated (p = 0.023, p = 0.011, and p = 0.027, respectively). In correlation analyses, the MMP-1 level showed a significant correlation with age (r = -0.48, p = 0.01,), and the MMP-9 level showed significant correlations with axial length (r = -0.59, p < 0.01) and DM duration (r = 049, p = 0.01). CONCLUSIONS: Concentrations of MMP-1 and MMP-9 were higher in the DME groups than in the control group. MMP-9 concentrations also differed depending on DR staging, DM duration, PRP treatment, and degree of axial myopia. MMP-9 may be more important than MMP-1 in the induction of DM complications in eyes.

Caspase-14 expression impairs retinal pigment epithelium barrier function: potential role in diabetic macular edema.

We recently showed that caspase-14 is a novel molecule in retina with potential role in accelerated vascular cell death during diabetic retinopathy (DR). Here, we evaluated whether caspase-14 is implicated in retinal pigment epithelial cells (RPE) dysfunction under hyperglycemia. The impact of high glucose (HG, 30 mM D-glucose) on caspase-14 expression in human RPE (ARPE-19) cells was tested, which showed significant increase in caspase-14 expression compared with normal glucose (5 mM D-glucose + 25 mM L-glucose). We also evaluated the impact of modulating caspase-14 expression on RPE cells barrier function, phagocytosis, and activation of other caspases using ARPE-19 cells transfected with caspase-14 plasmid or caspase-14 siRNA. We used FITC-dextran flux assay and electric cell substrate impedance sensing (ECIS) to test the changes in RPE cell barrier function. Similar to HG, caspase-14 expression in ARPE-19 cells increased FITC-dextran leakage through the confluent monolayer and decreased the transcellular electrical resistance (TER). These effects of HG were prevented by caspase-14 knockdown. Furthermore, caspase-14 knockdown prevented the HG-induced activation of caspase-1 and caspase-9, the only activated caspases by HG. Phagocytic activity was unaffected by caspase-14 expression. Our results suggest that caspase-14 contributes to RPE cell barrier disruption under hyperglycemic conditions and thus plays a role in the development of diabetic macular edema.

Pazopanib, a multitargeted tyrosine kinase inhibitor, reduces diabetic retinal vascular leukostasis and leakage.

OBJECTIVE: To determine the efficacy of pazopanib eye drops in the streptozotocin induced diabetic retinopathy rat model. METHODS: A 0.5% w/v pazopanib suspension was prepared in phosphate buffered saline (PBS, pH 7.4) in the presence of 0.5% w/v sodium carboxymethyl cellulose. Brown Norway rats were divided into three groups (n=4) - (1) healthy, (2) diabetic, and (3) diabetic with treatment. The drug suspension was administered twice daily as eye drops to group 3 for 30 days. Efficacy parameters including the number of adherent leukocytes in the retinal vasculature (leukostasis), blood-retinal FITC-dextran leakage, and vitreous-to-plasma protein ratio were measured. RESULTS: Pazopanib suspension in the form of eye drops significantly reduced leukostasis (32%), FITC-dextran leakage (39%), and the vitreous-to-plasma protein ratio (64%) in diabetic animals compared to untreated diabetic group. CONCLUSION: Pazopanib eye drops can alleviate retinal complications of diabetic retinopathy.

The relationship between plasma MMP-9 and TIMP-2 levels and intraocular pressure elevation in diabetic patients after intravitreal triamcinolone injection.

PURPOSE: To find out the relationship between steroid-induced intraocular pressure (IOP) rise and the plasma levels of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of MMP-2 (TIMP-2) in diabetic patients who underwent intravitreal triamcinolone acetonide (IVTA) injection for the treatment of diabetic macular edema. SUBJECTS AND METHODS: A total of 34 patients with diabetic macular edema who were treated with IVTA and 17 healthy subjects who served as control group for plasma MMP and TIMP levels were participated. Before IVTA treatment, patients and control subjects underwent complete ophthalmologic examination, including best-corrected visual acuity, slit-lamp biomicroscopy, IOP measurement with Goldmann applanation tonometry, and indirect ophthalmoscopy; and peripheral blood samples were collected from each study participants. Plasma levels of MMP-9, TIMP-2, and HbA1c levels were measured. Patients were seen 1, 6, 12, and 24 weeks after treatment and then every 6 months for up to 1 year for probable IOP rise. Patients were divided into 2 groups as having nonproliferative or proliferative diabetic retinopathy. These 2 groups were further classified according to their IOP levels as normal or elevated IOP (>21 mm Hg). RESULTS: The mean age of diabetic group of patients (n=34) and healthy control subjects (n=17) were 57.6+/-10.2 years (range: 22 to 70 y) and 53.1+/-10.3 years (range: 29 to 68 y), respectively. Seventeen (50%) diabetic patients had developed elevated IOP after a mean 2.2 months after IVTA injection. MMP-9 and TIMP-2 levels were found to be significantly higher in the diabetic groups with and without elevated IOP when compared with control group (P<0.001). MMP-9/TIMP-2 did not change significantly among the groups. Logistic regression analysis showed that only higher plasma TIMP-2 levels increase the risk of IOP elevation after IVTA injection in proliferative diabetic retinopathy (odds ratio=1.06, P<0.05). No significant relationship was found between IOP rise and HbA1c levels. CONCLUSIONS: The high levels of TIMP in diabetic patients might have a role on steroid-induced IOP rise. The key pathogenetic events that up-regulate TIMP levels should be investigated in steroid IOP rise in diabetics.

Association of manganese superoxide dismutase gene polymorphism (V16A) with diabetic macular edema in Korean type 2 diabetic patients.

This study was designed to investigate whether V16A polymorphism of the manganese superoxide dismutase (Mn-SOD) gene is associated with the development of type 2 diabetes mellitus and with progression of diabetic retinopathy (DR) and diabetic macular edema (DME). We simultaneously analyzed insertion/deletion polymorphism of the angiotensin-converting enzyme (ACE) gene in the 16th intron to avoid its confounding effect. A total of 192 nondiabetic subjects and 304 type 2 diabetic patients were included in the study. Diabetic retinopathy was classified as nonretinopathy, nonproliferative retinopathy, and proliferative retinopathy. Diabetic macular edema was defined as thickening of the retina and/or hard exudates within a 1-disk diameter of the center of the macula. Diabetic macular edema was further classified into focal, diffuse, and ischemic types. The A allele frequency of the Mn-SOD gene was not different between nondiabetic and type 2 diabetic subjects, between the normotensive and hypertensive groups, between the DR (-) and DR (+) groups, and among the stages of DR. In the DR (+) group, the DME (+) group had a lower A allele frequency than that of the DME (-) group. In the DME (+) group, focal, diffuse, and ischemic types were found in 8, 23, and 6 patients, respectively. The A allele frequency of each type was 0.188, 0.109, and 0.0. The D allele frequency of the angiotensin-converting enzyme gene did not differ in any of the comparisons. Clinical and laboratory parameters of the A allele carriers were not different from those of the noncarriers except for the prevalence of hypertension and DME. Hypertension, diabetic duration, and insulin therapy were related to DR. The A allele, hypertension, and insulin therapy were associated with DME. In conclusion, our results suggest that V16A polymorphism of the Mn-SOD gene is not related to the development of diabetes and progression of DR, but is associated with DME in Korean type 2 diabetic patients.

The potential role of PKC beta in diabetic retinopathy and macular edema.

Diabetic retinopathy is one of the most debilitating complications of diabetes mellitus. Despite major advances in understanding the pathogenesis of this disease and the efficacy of current therapies, diabetic retinopathy remains the leading cause of new-onset blindness among working-age people. The mainstay of current therapy, laser photocoagulation, is useful in preventing blindness and severe vision loss but is not often effective in restoring lost visual acuity. In addition, troublesome side effects and potentially serious complications may occur. Diabetic retinopathy is characterized by a progression of abnormalities. Nonproliferative retinopathy results from a series of biochemical and cellular changes that ultimately cause progressive retinal ischemia. The subsequent elaboration of growth factors in response to ischemia leads to the development of proliferative retinopathy, which is characterized by aberrant neovacularization of the retina-potentially leading to severe, irreversible visual loss. Increased retinal vascular leakage may also occur at any stage in this process, resulting in macular edema and possible progressive visual impairment. Although numerous biochemical factors are thought to play a role in the development of retinopathy, activation of protein kinase C (PKC), specifically the beta isoform of PKC (PKC beta), is implicated for both the early and late-stage manifestations of retinopathy. Studies suggest that orally administered LY333531, a beta-isoform specific PKC inhibitor, may be effective in ameliorating retinopathy progression, proliferation, and retinal vascular leakage. The status of ongoing clinical trials aimed at addressing the efficacy of PKC beta with regard to diabetes-induced retinal complications and perspectives on the role of PKC beta are presented.

Inhibition of membrane-bound carbonic anhydrase decreases subretinal pH and volume.

PURPOSE: The lipophilic carbonic anhydrase (CA) inhibitor acetazolamide has been shown to enhance subretinal fluid resorption, reduce subretinal pH, and can improve cystoid macular edema, but its clinical use is limited by systemic side effects. While these are most likely a result of inhibiting intracellular CA isoenzymes, retinal pigment epithelial (RPE) transport is thought to be modulated via membrane-bound CA. This study investigates whether benzolamide, a hydrophilic CA inhibitor that does not readily penetrate cell membranes, is sufficient to modulate subretinal volume and pH. METHODS: Volume and pH were assessed in the subretinal space (SRS) of the perfused chick retina-RPE-choroid preparation by calculating these variables from data obtained with two different double-barreled, ion-selective electrodes (H+ for pH and the extracellular space marker tetramethylammonium (TMA+) for SRS volume). Light induced variations and changes in baseline measurements were recorded before and after addition of 10(-4) M acetazolamide or benzolamide to the basal perfusion. RESULTS: Basal perfusion with either drug induced both an acidification of the SRS by 0.02-0.04 pH units, which occurred within 60 s, as well as an increase in the amplitude of the light-induced alkalinisation of the SRS. TMA+ concentration in the SRS increased steadily over a period of several minutes after basal perfusion with either of the CA inhibitors, and the calculated SRS volume was reduced by 40% within 8-10 min. CONCLUSION: The observation that benzolamide had effects equal to acetazolamide suggests that inhibition of membrane-bound CA at the basolateral membrane of the RPE is sufficient to decrease subretinal pH and volume. This may represent a clinically important mechanism for the resorption of sub- and intraretinal fluid.

The role of carbonic anhydrase inhibitors in the management of macular edema.

Medical treatment of cystoid macular edema (CME) with carbonic anhydrase inhibitors has been known for over a decade. Initial observations were based on experimental data which suggested that acetazolamide can increase fluid absorption across the retinal pigment epithelium. Carbonic anhydrase inhibitors (CAI) have also been shown to have other direct effects both on retinal and retinal pigment epithelial cell function by inducing an acidification of the subretinal space, a decrease of the standing potential as well as an increase in retinal adhesiveness. It is thought that acidification of the subretinal space is finally responsible for the increase in fluid resorption from the retina through the RPE into the choroid. Several clinical studies have suggested that patients with cystoid macular edema due to retinitis pigmentosa and uveitis may react more favorably to CAI treatment than other etiologies such as diabetic maculopathy or macular edema after retinal vein occlusion. The present working hypothesis is that diffuse leakage from the RPE responds more readily to CAI treatment than leakage from retinal vessels. This may be due to the modulation of membrane- bound CA IV in the RPE which may have lost its polarised distribution in the presence of macular edema. A normal clinical starting dose of CAI is 500 mg/day which should be continued for at least one month to see an effect. This dose may be reduced by the patients over the course of therapy. Metaphylaxis to the drug may occur with a rebound of the edema despite continuation of treatment.