Rangasamy Sampathkumar, McGuire Paul G, Franco Nitta Carolina, Monickaraj Finny, Oruganti Sreenivasa R, Das Arup
Department of Cell Biology & Physiology, University of New Mexico School of Medicine, Albuquerque, New Mexico, United States of America.
Department of Surgery, University of New Mexico School of Medicine, Albuquerque, New Mexico, United States of America; New Mexico VA Health Care System, Albuquerque, New Mexico, United States of America.
PLoS One. 2014 Oct 20;9(10):e108508. doi: 10.1371/journal.pone.0108508. eCollection 2014.
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.
糖尿病视网膜中的炎症是由白细胞粘附于视网膜血管系统以及血视网膜屏障（BRB）改变介导的。我们研究了趋化因子在糖尿病患者BRB改变中的作用。通过注射链脲佐菌素使动物患糖尿病，并分析其基因表达和单核细胞/巨噬细胞浸润情况。在糖尿病4周和8周的大鼠视网膜中，以及在高糖和葡萄糖通量处理的人视网膜内皮细胞中，CCL2（趋化因子配体2）的表达均显著上调。此外，糖尿病或眼内注射重组CCL2导致巨噬细胞标志物F4/80的表达增加。细胞培养阻抗传感研究表明，纯化的CCL2无法改变人视网膜内皮细胞屏障的完整性，而单核细胞条件培养基导致细胞电阻显著降低，这表明CCL2在早期免疫细胞募集中与随后的屏障改变相关。此外，使用Cx3cr1-GFP小鼠，我们发现眼内注射CCL2会增加视网膜GFP+单核细胞/巨噬细胞浸润。当这些小鼠患糖尿病时，视网膜组织中也存在单核细胞/巨噬细胞浸润增加的情况。糖尿病和CCL2注射还诱导了这些动物视网膜小胶质细胞的激活。通过流式细胞术定量分析表明，与对照非糖尿病动物相比，野生型糖尿病动物视网膜中CX3CR1+/CD11b+（单核细胞/巨噬细胞和小胶质细胞）细胞增加了两倍。使用CCL2基因敲除（Ccl2-/-）小鼠，我们发现糖尿病诱导后视网膜血管渗漏和单核细胞浸润显著减少，这表明这种趋化因子在BRB改变中具有重要作用。因此，CCL2可能是治疗糖尿病性黄斑水肿的重要治疗靶点。
