Peripheral Blood Mononuclear Cells from Patients with Type 1 Diabetes and Diabetic Retinopathy Produce Higher Levels of IL-17A, IL-10 and IL-6 and Lower Levels of IFN-γ-A Pilot Study.

Inflammation is key to the pathogenesis of diabetic retinopathy (DR). This prospective study investigated alterations in inflammatory cytokines in peripheral blood mononuclear cells (PBMCs) in 41 people with type 1 diabetes (T1D), sub-grouped into mild non-proliferative DR (mNPDR; n = 13) and active and inactive (each n = 14) PDR. Age/gender-matched healthy controls (n = 13) were included. PBMCs were isolated from blood samples. Intracellular cytokine expression by PBMCs after 16-h stimulation (either E. coli lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate plus ionomycin, D-glucose or D-mannitol) were assessed by flow cytometry. Cytokine production in plasma, non-stimulated and LPS-stimulated PBMC supernatant was also assessed. Increased BMC IL-10 secretion and reduced expression of IL-6 and IFN-γ in CD3+ cells were observed in mNPDR. Reduced IL-6 and IL-10 secretion, and higher levels of intracellular IL-6 expression, especially in CD11b+ PBMCs, was detected in aPDR; levels were positively correlated with DR duration. Patients with T1D demonstrated increased intracellular expression of IL-17A in myeloid cells and reduced IFN-γ expression in CD3+ cells. Plasma levels of IL-1R1 were increased in mNPDR compared with controls. Results suggest that elevated PBMC-released IL-10, IL-6, in particular myeloid-produced IL-17A, may be involved in early stages of DR. IL-6-producing myeloid cells may play a role in PDR development.

Hyaloid Vasculature as a Major Source of STAT3+ (Signal Transducer and Activator of Transcription 3) Myeloid Cells for Pathogenic Retinal Neovascularization in Oxygen-Induced Retinopathy.

OBJECTIVE: Myeloid cells are critically involved in inflammation-induced angiogenesis, although their pathogenic role in the ischemic retina remains controversial. We hypothesize that myeloid cells contribute to pathogenic neovascularization in retinopathy of prematurity through STAT3 (signal transducer and activator of transcription 3) activation. Approach and Results: Using the mouse model of oxygen-induced retinopathy, we show that myeloid cells (CD45+IsolectinB4 [IB4]+) and particularly M2-type macrophages (CD45+ Arg1+), comprise a major source of STAT3 activation (pSTAT3) in the immature ischemic retina. Most of the pSTAT3-expressing myeloid cells concentrated at the hyaloid vasculature and their numbers were strongly correlated with the severity of pathogenic neovascular tuft formation. Pharmacological inhibition of STAT3 reduced the load of IB4+ cells in the hyaloid vasculature and significantly reduced the formation of pathogenic neovascular tufts during oxygen-induced retinopathy, leading to improved long-term visual outcomes (ie, increased retinal thickness and scotopic b-wave electroretinogram responses). Genetic deletion of SOCS3 (suppressor of cytokine signaling 3), an endogenous inhibitor of STAT3, in myeloid cells, enhanced pathological and physiological neovascularization in oxygen-induced retinopathy, indicating that myeloid-STAT3 signaling is crucial for retinal angiogenesis. CONCLUSIONS: Circulating myeloid cells may migrate to the immature ischemic retina through the hyaloid vasculature and contribute to retinal neovascularization via activation of STAT3. Understanding how STAT3 modulates myeloid cells for vascular repair/pathology may provide novel therapeutic options in pathogenic angiogenesis.

Circulating Leukocyte Alterations and the Development/Progression of Diabetic Retinopathy in Type 1 Diabetic Patients - A Pilot Study.

BACKGROUND/AIMS: The aim of this study was to investigate the relationship between alterations in circulating leukocytes and the initiation and progression of DR in people with type 1 diabetes (T1D). METHODS: Forty-one patients with T1D [13 mild non-proliferative DR (mNPDR), 14 active proliferative DR (aPDR) and 14 inactive PDR (iPDR)], and 13 age- and gender-matched healthy controls were recruited prospectively. Circulating leukocytes, including CD4+ and CD8+ T-cells, CD14+CD16-, CD14-CD16+ and CD14+CD16+ monocytes; CD16+HLA-DR- neutrophils, CD19+ B-cells and CD56+ natural killer cells and their cell surface adhesion molecules and chemokine receptors (HLA-DR, CD62L, CCR2, CCR5, CD66a, CD157 and CD305) were examined by flow cytometry. RESULTS: In DR patients, compared to healthy controls, increased proportions of neutrophils (p = .0152); reduced proportions of lymphocytes (p = .0002), HLA-DR+ leukocytes (p = .0406) and non-classical monocytes (p = .0204); and reduced expression of CD66a (p = .0048) and CD157 (p = .0007) on CD4+ T cells were observed. Compared to healthy controls, CD19+ B cells were reduced at the mNPDR but not aPDR patients. Total lymphocytes, CD4+ T cells and CD8+ T cells progressively decreased whereas neutrophils, the neutrophil/lymphocyte ratio and the neutrophil/CD4+ ratio progressively increased from early to late stages of DR, reaching statistical significance at the aPDR stage. Longer diabetes duration was associated with a reduced proportion of CD8+ T cells (p = .002) and increased neutrophil/CD8+ ratio (p = .033). CONCLUSIONS: In this pilot study, DR is associated with increased innate cellular immunity especially neutrophils and reduced adaptive cellular immunity particularly lymphocytes. Impaired B-cell immunity may play a role in the initiation of DR; whereas impaired T-cell immunity with increased neutrophil response may contribute to progression of DR from non-proliferative to proliferative stages in T1D patients. Large multicenter studies are needed to further understand the immune dysregulation in DR initiation and progression.

Cytokine Signaling Protein 3 Deficiency in Myeloid Cells Promotes Retinal Degeneration and Angiogenesis through Arginase-1 Up-Regulation in Experimental Autoimmune Uveoretinitis.

The suppressor of cytokine signaling protein 3 (SOCS3) critically controls immune cell activation, although its role in macrophage polarization and function remains controversial. Using experimental autoimmune uveoretinitis (EAU) as a model, we show that inflammation-mediated retinal degeneration is exaggerated and retinal angiogenesis is accelerated in mice with SOCS3 deficiency in myeloid cells (LysMCre/+SOCS3fl/fl). At the acute stage of EAU, the population of infiltrating neutrophils was increased and the population of macrophages decreased in LysMCre/+SOCS3fl/fl mice compared with that in wild-type (WT) mice. Real-time RT-PCR showed that the expression of tumor necrosis factor-α, IL-1ß, interferon-γ, granulocyte-macrophage colony-stimulating factor, and arginase-1 was significantly higher in the LysMCre/+SOCS3fl/fl EAU retina in contrast to the WT EAU retina. The percentage of arginase-1+ infiltrating cells was significantly higher in the LysMCre/+SOCS3fl/fl EAU retina than that in the WT EAU retina. In addition, bone marrow-derived macrophages and neutrophils from the LysMCre/+SOCS3fl/fl mice express significantly higher levels of chemokine (C-C motif) ligand 2 and arginase-1 compared with those from WT mice. Inhibition of arginase using an l-arginine analog amino-2-borono-6-hexanoic suppressed inflammation-induced retinal angiogenesis without affecting the severity of inflammation. Our results suggest that SOCS3 critically controls the phenotype and function of macrophages and neutrophils under inflammatory conditions and loss of SOCS3 promotes the angiogenic phenotype of the cells through up-regulation of arginase-1.

Topical Delivery of Anti-VEGF Drugs to the Ocular Posterior Segment Using Cell-Penetrating Peptides.

Purpose: To evaluate the efficacy of anti-VEGF agents for treating choroidal neovascularization (CNV) when delivered topically using novel cell-penetrating peptides (CPPs) compared with delivery by intravitreal (ivit) injection. Methods: CPP toxicity was investigated in cell cultures. Ivit concentrations of ranibizumab and bevacizumab after topical administration were measured using ELISA. The biological efficacy of topical anti-VEGF + CPP complexes was compared with ivit anti-VEGF injections using an established model of CNV. Results: CPPs were nontoxic in vitro. In vivo, after topical eye drop delivery, CPPs were present in the rat anterior chamber within 6 minutes. A single application of CPP + bevacizumab eye drop delivered clinically relevant concentrations of bevacizumab to the posterior chamber of the rat eye in vivo. Similarly, clinically relevant levels of CPP + ranibizumab and CPP + bevacizumab were detected in the porcine vitreous and retina ex vivo. In an established model of CNV, mice treated with either a single ivit injection of anti-VEGF, twice daily CPP + anti-VEGF eye drops or daily dexamethasone gavage for 10 days all had significantly reduced areas of CNV when compared with lasered eyes without treatment. Conclusions: CPPs are nontoxic to ocular cells and can be used to deliver therapeutically relevant doses of ranibizumab and bevacizumab by eye drop to the posterior segment of mouse, rat, and pig eyes. The CPP + anti-VEGF drug complexes were cleared from the retina within 24 hours, suggesting a daily eye drop dosing regimen. Daily, topically delivered anti-VEGF with CPP was as efficacious as a single ivit injection of anti-VEGF in reducing areas of CNV in vivo.

No evidence for loss of short-wavelength sensitive cone photoreceptors in normal ageing of the primate retina.

In old world primates including humans, cone photoreceptors are classified according to their maximal sensitivity at either short (S, blue), middle (M, green) or long (L, red) wavelengths. Colour discrimination studies show that the S-cone pathway is selectively affected by age and disease, and psychophysical models implicate their loss. Photoreceptors have high metabolic demand and are susceptible to age or disease-related losses in oxygen and nutrient supply. Hence 30% of rods are lost over life. While comparable losses are not seen in cones, S-cones comprise less than 10% of the cone population, so significant loss would be undetected in total counts. Here we examine young and aged primate retinae stained to distinguish S from M/L-cones. We show there is no age-related cone loss in either cone type and that S-cones are as regularly distributed in old as young primates. We propose that S-cone metabolism is less flexible than in their M/L counterparts, making them more susceptible to deficits in normal cellular function. Hypoxia is a feature of the ageing retina as extracellular debris accumulates between photoreceptors and their blood supply which likely impacts S-cone function. However, that these cells remain in the ageing retina suggests the potential for functional restoration.

Topical cyclodextrin reduces amyloid beta and inflammation improving retinal function in ageing mice.

Retinal ageing results in chronic inflammation, extracellular deposition, including that of amyloid beta (Aß) and declining visual function. In humans this can progress into age-related macular degeneration (AMD), which is without cure. Therapeutic approaches have focused on systemic immunotherapies without clinical resolution. Here, we show using aged mice that 2-Hydroxypropyl-ß-cyclodextrin, a sugar molecule given as eye drops over 3 months results in significant reductions in Aß by 65% and inflammation by 75% in the aged mouse retina. It also elevates retinal pigment epithelium specific protein 65 (RPE65), a key molecule in the visual cycle, in aged retina. These changes are accompanied by a significant improvement in retinal function measured physiologically. 2-Hydroxypropyl-ß-cyclodextrin is as effective in reducing Aß and inflammation in the complement factor H knockout (Cfh(-/-)) mouse that shows advanced ageing and has been proposed as an AMD model. ß-cyclodextrin is economic, safe and may provide an efficient route to reducing the impact of retinal ageing.

Comparison of stability, cellular, glucose-lowering and appetite supressing effects of oxyntomodulin analogues modified at the N-terminus.

Oxyntomodulin (Oxm) possesses beneficial biological actions for the potential treatment of obesity-diabetes. However, rapid inactivation by dipeptidyl peptidase-4 (DPP-4) results in a short half-life, hindering therapeutic applicability. In the present study, six Oxm analogues namely, (Thr(2))Oxm, (Asp(3))Oxm, (Aib(2))Oxm, (d-Ser(2))Oxm, (N-acetyl)Oxm and (d-Ser(2))Oxm-Lys-γ-glutamyl-PAL were synthesised and tested for DPP-4 stability and biological activity. Native Oxm, (Thr(2))Oxm and (Asp(3))Oxm were rapidly degraded by DPP-4, while (Aib(2))Oxm, (d-Ser(2))Oxm, (N-acetyl)Oxm and (d-Ser(2))Oxm-Lys-γ-glutamyl-PAL were resistant to degradation. All peptides stimulated cAMP production (P<0.01 to P<0.001) in GLP-1-R, but not in GIP-R, transfected cells. In glucagon-R transfected cells, all peptides except (N-acetyl)Oxm and (Thr(2))Oxm evoked significant cAMP generation. Similarly, all analogues, except (N-acetyl)Oxm, exhibited prominent (P<0.05 to P<0.001) insulinotropic activity in BRIN BD11 cells. When administered in conjunction with glucose to normal mice only native Oxm, (Aib(2))Oxm and (d-Ser(2))Oxm significantly (P<0.05 to P<0.01) increased overall plasma insulin levels. The corresponding glycaemic excursion was significantly (P<0.05 to P<0.001) lowered by all Oxm peptides, barring (N-acetyl)Oxm. Further investigations revealed persistent glucose-lowering and insulin-releasing actions of (d-Ser(2))Oxm-Lys-γ-glutamyl-PAL. Studies in GIP- and GLP-1-receptor KO mice with (Aib(2))Oxm, (d-Ser(2))Oxm, and (d-Ser(2))Oxm-Lys-γ-glutamyl-PAL highlighted the importance of GLP-1 receptor signalling for the beneficial glucose homoeostatic actions of these analogues. All peptides, except (N-acetyl)Oxm, possessed significant appetite suppressive effects in mice. These data highlight the significant therapeutic promise of enzymatically stable Oxm-based peptides, particularly with position 2 modifications, for the treatment of obesity-diabetes.

A novel DPP IV-resistant C-terminally extended glucagon analogue exhibits weight-lowering and diabetes-protective effects in high-fat-fed mice mediated through glucagon and GLP-1 receptor activation.

AIMS/HYPOTHESIS: Modification of the structure of glucagon could provide useful compounds for the potential treatment of obesity-related diabetes. METHODS: This study evaluated N-acetyl-glucagon, (D-Ser(2))glucagon and an analogue of (D-Ser(2))glucagon with the addition of nine amino acids from the C-terminal of exendin(1-39), namely (D-Ser(2))glucagon-exe. RESULTS: All analogues were resistant to dipeptidyl peptidase IV degradation. N-Acetyl-glucagon lacked acute insulinotropic effects in BRIN BD11 cells, whereas (D-Ser(2))glucagon and (D-Ser(2))glucagon-exe evoked significant (p < 0.001) insulin release. (D-Ser(2))glucagon-exe stimulated cAMP production (p < 0.001) in glucagon- and GLP-1-receptor (GLP-1R)-transfected cells but not in glucose-dependent insulinotropic polypeptide-receptor-transfected cells. In normal mice, N-acetyl-glucagon and (D-Ser(2))glucagon retained glucagon-like effects of increasing (p < 0.001) plasma glucose and insulin levels. (D-Ser(2))glucagon-exe was devoid of hyperglycaemic actions but substantially (p < 0.001) increased plasma insulin levels. (D-Ser(2))glucagon-exe reduced the glycaemic excursion (p < 0.01) and increased the insulin secretory (p < 0.01) response following a glucose challenge 12 h after administration. Studies in GLP-1R knockout mice confirmed involvement of the GLP-1R pathway in the biological actions of (D-Ser(2))glucagon-exe. Twice-daily administration of (D-Ser(2))glucagon-exe to high-fat-fed mice for 28 days significantly (p < 0.05 to p < 0.001) reduced body weight, energy intake and non-fasting glucose levels, as well as increasing insulin concentrations. Glucose tolerance and insulin sensitivity were significantly (p < 0.01) improved and energy expenditure, O2 consumption and locomotor activity were (p < 0.05 to p < 0.001) augmented. The metabolic benefits were accompanied by increases in pancreatic islet number (p < 0.001) and area (p < 0.05), as well as beta cell area (p < 0.05). Beneficial effects were largely retained for 14 days following cessation of treatment. CONCLUSIONS/INTERPRETATION: This study emphasises the potential of (D-Ser(2))glucagon-exe for the treatment of obesity-related diabetes.