a-simplified-method-for-isolation-culture-retinal-pigment-epithelial

A Simplified Method for Isolation and Culture of Retinal Pigment Epithelial Cells from Adult Mice

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important ...

Case Western Reserve University

CD40 expressed in Müller cells is a central driver of diabetic retinopathy. CD40 causes phospholipase Cγ1 (PLCγ1)-dependent ATP release in Müller cells followed by purinergic receptor (P2X)-dependent production of proinflammatory cytokines in myeloid cells. In the diabetic retina, CD40 and P2X upregulate a broad range of inflammatory molecules that promote development of diabetic retinopathy. The molecular event downstream of CD40 that activates the PLCγ1-ATP-P2X-proinflammatory cytokine cascade and promotes development of diabetic retinopathy is unknown. We hypothesise that disruption of the CD40-driven molecular events that trigger this cascade prevents/treats diabetic retinopathy in mice.

Disruption of retinal inflammation and the development of diabetic retinopathy in mice by a CD40-derived peptide or mutation of CD40 in Müller cells.

CD40 is upregulated in the retinas of diabetic mice, drives pro-inflammatory molecule expression, and promotes diabetic retinopathy. The role of CD40 in diabetic retinopathy in humans is unknown. Upregulation of CD40 and its downstream signaling molecules TNF receptor associated factors (TRAFs) is a key feature of CD40-driven inflammatory disorders. We examined the expression of CD40, TRAF2, and TRAF6 as well as pro-inflammatory molecules in retinas from patients with diabetic retinopathy.

CD40 Upregulation in the Retina of Patients With Diabetic Retinopathy: Association With TRAF2/TRAF6 Upregulation and Inflammatory Molecule Expression.

CD40 induces pro-inflammatory responses in endothelial and Müller cells and is required for the development of diabetic retinopathy (DR). CD40 is upregulated in these cells in patients with DR. CD40 upregulation is a central feature of CD40-driven inflammatory disorders. What drives CD40 upregulation in the diabetic retina remains unknown. We examined the role of advanced glycation end products (AGEs) in CD40 upregulation in endothelial cells and Müller cells. Human endothelial cells and Müller cells were incubated with unmodified or methylglyoxal (MGO)-modified fibronectin. CD40 expression was assessed by flow cytometry. The expression of ICAM-1 and CCL2 was examined by flow cytometry or ELISA after stimulation with CD154 (CD40 ligand). The expression of carboxymethyl lysine (CML), fibronectin, and laminin as well as CD40 in endothelial and Müller cells from patients with DR was examined by confocal microscopy. Fibronectin modified by MGO upregulated CD40 in endothelial and Müller cells. CD40 upregulation was functionally relevant. MGO-modified fibronectin enhanced CD154-driven upregulation of ICAM-1 and CCL2 in endothelial and Müller cells. Increased CD40 expression in endothelial and Müller cells from patients with DR was associated with increased CML expression in fibronectin and laminin. These findings identify AGEs as inducers of CD40 upregulation in endothelial and Müller cells and enhancers of CD40-dependent pro-inflammatory responses. CD40 upregulation in these cells is associated with higher CML expression in fibronectin and laminin in patients with DR. This study revealed that CD40 and AGEs, two important drivers of DR, are interconnected.

Sarah Vos

Division of Infectious Diseases and HIV Medicine,
Department of Medicine,
Division of Infectious Diseases and HIV Medicine, Department of Medicine

Case Western Reserve University

A Simplified Method for Isolation and Culture of Retinal Pigment Epithelial Cells from Adult Mice

Sarah Vos

Division of Infectious Diseases and HIV Medicine,Department of Medicine,Division of Infectious Diseases and HIV Medicine, Department of Medicine

Case Western Reserve University

A Simplified Method for Isolation and Culture of Retinal Pigment Epithelial Cells from Adult Mice

Division of Infectious Diseases and HIV Medicine,
Department of Medicine,
Division of Infectious Diseases and HIV Medicine, Department of Medicine