Carolina Franco Nitta

Chemokine Mediated Monocyte Trafficking into the Retina: Role of Inflammation in Alteration of the Blood-Retinal Barrier in Diabetic Retinopathy

Inflammation in the diabetic retina is mediated by leukocyte adhesion to the retinal vasculature and alteration of the blood-retinal barrier (BRB). We investigated the role of chemokines in the alteration of the BRB in diabetes. Animals were made diabetic by streptozotocin injection and analyzed for gene expression and monocyte/macrophage infiltration. The expression of CCL2 (chemokine ligand 2) was significantly up-regulated in the retinas of rats with 4 and 8 weeks of diabetes and also in human retinal endothelial cells treated with high glucose and glucose flux. Additionally, diabetes or intraocular injection of recombinant CCL2 resulted in increased expression of the macrophage marker, F4/80. Cell culture impedance sensing studies showed that purified CCL2 was unable to alter the integrity of the human retinal endothelial cell barrier, whereas monocyte conditioned medium resulted in significant reduction in cell resistance, suggesting the relevance of CCL2 in early immune cell recruitment for subsequent barrier alterations. Further, using Cx3cr1-GFP mice, we found that intraocular injection of CCL2 increased retinal GFP+ monocyte/macrophage infiltration. When these mice were made diabetic, increased infiltration of monocytes/macrophages was also present in retinal tissues. Diabetes and CCL2 injection also induced activation of retinal microglia in these animals. Quantification by flow cytometry demonstrated a two-fold increase of CX3CR1+/CD11b+ (monocyte/macrophage and microglia) cells in retinas of wildtype diabetic animals in comparison to control non-diabetic ones. Using CCL2 knockout (Ccl2−/−) mice, we show a significant reduction in retinal vascular leakage and monocyte infiltration following induction of diabetes indicating the importance of this chemokine in alteration of the BRB. Thus, CCL2 may be an important therapeutic target for the treatment of diabetic macular edema.

Basement membrane stiffening promotes retinal endothelial activation associated with diabetes

Endothelial activation is a hallmark of the high‐glucose (HG)‐induced retinal inflammation associated with diabetic retinopathy (DR). However, precisely how HG induces retinal endothelial activation is not fully understood. We hypothesized that HG‐induced upregulation of lysyl oxidase (LOX), a collagen‐cross‐linking enzyme, in retinal capillary endothelial cells (ECs) enhances subendothelial basement membrane (BM) stiffness, which, in turn, promotes retinal EC activation. Diabetic C57BL/6 mice exhibiting a 70 and 50% increase in retinal intercellular adhesion molecule (ICAM)‐1 expression and leukocyte accumulation, respectively, demonstrated a 2‐fold increase in the levels of BM collagen IV and LOX, key determinants of capillary BM stiffness. Using atomic force microscopy, we confirmed that HG significantly enhances LOX‐dependent subendothelial matrix stiffness invitro, which correlated with an ~2.5‐fold increase in endothelial ICAM‐1 expression, a 4‐fold greater monocyte—EC adhesion, and an ~ 2‐fold alteration in endothelial NO (decrease) and NF‐κB activation (increase). Inhibition of LOX‐dependent subendothelial matrix stiffening alone suppressed HG‐induced retinal EC activation. Finally, using synthetic matrices of tunable stiffness, we demonstrated that subendothelial matrix stiffening is necessary and sufficient to promote EC activation. These findings implicate BM stiffening as a critical determinant of HG‐induced retinal EC activation and provide a rationale for examining BM stiffness and underlying mechanotransduction pathways as therapeutic targets for diabetic retinopathy.—Yang, X., Scott, H. A., Monickaraj, F., Xu, J., Ardekani, S., Nitta, C. F., Cabrera, A., McGuire, P. G., Mohideen, U., Das, A., Ghosh, K. Basement membrane stiffening promotes retinal endothelial activation associated with diabetes. FASEB J. 30, 601‐611 (2016). www.fasebj.org

Crosstalk between Immune Cells and Adipocytes Requires Both Paracrine Factors and Cell Contact to Modify Cytokine Secretion

Increased adiposity results in a heightened infiltration of immune cells into fat depots, which in turn generates a pro-inflammatory phenotype in obese individuals. To better understand the causal factors that establish this pro-inflammatory profile, we examined events leading to crosstalk between adipocytes and immune cells. Using isolated spleen-derived immune cells, stimulated with LPS, together with cultured adipocytes, we differentiated the effects of paracrine factors and cell-cell contact on TNFα, IL-6 and MCP-1 secretion levels and secretion profiles. When splenocytes and adipocytes were co-cultured without direct contact, permitting only paracrine communication, secretion of IL-6 and MCP-1 were increased by 3- and 2.5-fold, respectively, over what was secreted by individual cultures, whereas TNFα secretion was reduced by 55%. When cells were co-cultured with direct cell-cell contact, IL-6 and MCP-1 secretion were increased by an additional 36% and 38%, respectively, over that measured from just paracrine stimulation alone, indicating that cell contact provides a synergistic signal that amplifies elevated cytokine secretion stimulated by paracrine signals. Using splenocytes from TNFα-/- mice showed that the absence of TNFα has little effect on paracrine stimulation of cytokine secretion, but attenuates cell contact-mediated enhancement of IL-6 and MCP-1 secretion. Furthermore, TNFα supports cell contact-mediated signaling in part, but not exclusively, through Nuclear Factor-κB activation. These findings indicate that engagement of cell contact between immune cells and adipocytes, in conjunction with locally secreted paracrine factors, activates a unique signaling pathway that mediates crosstalk between these cell types leading to marked effects on cytokine secretion and profile.

Cathepsin D: an Mϕ-derived factor mediating increased endothelial cell permeability with implications for alteration of the blood-retinal barrier in diabetic retinopathy

Inflammation plays an important role in the pathogenesis of diabetic retinopathy (DR). We have previously reported increased monocyte (Mono) trafficking into the retinas of diabetic animals. In this study, we have examined the effect of activated Monos on retinal endothelial cells (ECs). The U937 Mφ‐conditioned medium (CM) significantly decreased the transendothelial resistance of EC monolayers as measured by electric cell‐substrate impedance sensing (P = 0.007). The CM was fractioned, and the effective fraction (30–100 kDa) was analyzed by liquid chromatography‐mass spectrometry, and cathepsin D (CD) was identified as a major secreted product. Immunoprecipitated CD resulted in decreased resistance in ECs (P = 0.006). The specificity of CD in mediating alterations of the EC barrier was confirmed using small interfering RNA. The decreased resistance correlated with a significantly increased gap between ECs. CD altered the Ras homolog gene family, member A/Rho‐associated kinase pathway with increased stress actin filament formation in the EC layer. Increased CD levels were found in the retinas of diabetic mice (3‐fold) and serum samples of patients with diabetic macular edema (1.6‐fold) measured by Western blot and ELISA. These findings suggest an important role for Mφ‐derived CD in altering the blood‐retinal barrier and reveal a potential therapeutic target in the treatment of DR.—Monickaraj, F., McGuire, P. G., Nitta, C. F., Ghosh, K., Das, A. Cathepsin D: an Mφ‐derived factor mediating increased endothelial cell permeability with implications for alteration of the blood‐retinal barrier in diabetic retinopathy. FASEB J. 30, 1670–1682 (2016). www.fasebj.org