Pablo F. Barcelona

Activated α2-macroglobulin induces Müller glial cell migration by regulating MT1-MMP activity through LRP1

In retinal proliferative diseases, Müller glial cells (MGCs) acquire migratory abilities. However, the mechanisms that regulate this migration remain poorly understood. In addition, proliferative disorders associated with enhanced activities of matrix metalloprotease 2 (MMP‐2) and MMP‐9 also present increased levels of the protease inhibitor α2‐macroglobulin (α2M) and its receptor, the low‐density lipoprotein receptor‐related protein 1 (LRP1). In the present work, we investigated whether the protease activated form of α2M, α2M∗, and LRP1 are involved with the MGC migratory process. By performing wound‐scratch migration and zymography assays, we demonstrated that α2M∗ induced cell migration and proMMP‐2 activation in the human Müller glial cell line, MIO‐M1. This induction was blocked when LRP1 and MT1‐MMP were knocked down with siRNA techniques. Using fluorescence microscopy and biochemical procedures, we found that α2M∗ induced an increase in LRP1 and MT1‐MMP accumulation in early endosomes, followed by endocytic recycling and intracellular distribution of MT1‐MMP toward cellular protrusions. Moreover, Rab11‐dominant negative mutant abrogated MT1‐MMP recycling pathway, cell migration, and proMMP‐2 activation induced by α2M∗. In conclusion, α2M∗, through its receptor LRP1, induces cellular migration of Müller glial cells by a mechanism that involves MT1‐MMP intracellular traffic to the plasma membrane by a Rab11‐dependent recycling pathway.—Barcelona, P. F., Jaldín‐Fincati, J. R., Sánchez, M. C.Chiabrando, G. A., Activated α2‐macroglobulin induces Müller glial cell migration by regulating MT1‐MMP activity through LRP1. FASEBJ. 27, 3181‐3197 (2013). www.fasebj.org

Immunohistochemical localization of low density lipoprotein receptor-related protein 1 and α2-Macroglobulin in retinal and choroidal tissue of proliferative retinopathies

The immunolocalization of the low density lipoprotein receptor-related protein 1 (LRP1) and its ligand alpha 2-Macroglobulin (alpha(2)M) was examined in tissues from human donor eyes of normal, diabetic and sickle cell disease subjects. Streptavidin alkaline phosphatase immunohistochemistry was performed with a mouse anti-human LRP1 and rabbit anti-human alpha(2)M antibodies. Retinal and choroidal blood vessels were labeled with mouse anti-human CD34 antibody in adjacent tissue sections. Mean scores for immunostaining from the pathological and control eyes were statistically compared. LRP1 immunoreactivity was very weak to negative in the neural retina of normal subjects except in scattered astrocytes. LRP1 expression in diabetic eyes was detected in the internal limiting membrane (ILM), astrocytes, inner photoreceptor matrix, choriocapillaris and choroidal stroma. The ligand alpha(2)M, however, was limited mainly to blood vessel walls, some areas of the inner nuclear layer (INL), photoreceptors, RPE-Bruchs membrane-choriocapillaris complex, intercapillary septa, and choroidal stroma. In sickle cell eyes, avascular and vascular retina as well as choroidal neovascularization (CNV) were analyzed. In avascular areas, LRP1 immunoreactivity was in innermost retina (presumably ILM, astrocytes, and Muller cells) and INL as well as RPE-Bruchs membrane-choriocapillaris complex and choroidal stroma. alpha(2)M was very weak in avascular peripheral retina compared to vascularized areas and limited to stroma in choroid. In contrast, in areas with CNV, LRP1 immunoreactivity was significantly decreased in overlying retina and in RPE-Bruchs membrane and choroidal stroma compared to the controls, while alpha(2)M was elevated in RPE-Bruchs membrane near CNV compared to normal areas in sickle cell choroid. The mean scores revealed that LRP1 and alpha(2)M in neural retina were significantly elevated in astrocytes and ILM in diabetic eyes (p < or = 0.05), whereas in sickle cell eyes scores were elevated in ILM and INL (p < or = 0.05). In addition, alpha(2)M immunoreactivity was in photoreceptors in both ischemic retinopathies. In choroid, the patterns of LRP1 and alpha(2)M expression were different and not coincident. This is the first demonstration of the presence of LRP1 and alpha(2)M in human proliferative retinopathies. Elevated LRP1 expression in sickle cell neural retina and diabetic inner retina and choroid suggests that LRP1 plays an important role in ischemic neovascular diseases.

α2-Macroglobulin Induces Glial Fibrillary Acidic Protein Expression Mediated by Low-Density Lipoprotein Receptor-Related Protein 1 in Müller Cells

PURPOSE Although it is known that Müller cells express the glial fibrillary acidic protein (GFAP) in response to acute retinal damage, the regulatory mechanism is not completely understood. α(2)-Macroglobulin (α(2)M) and its receptor, low-density lipoprotein receptor-related protein 1 (LRP1), have also been found in injured retinas. Herein, the authors examined the involvement of the α(2)M/LRP1 system in GFAP expression in Müller cells using in vitro and in vivo experimental models. METHODS Using Western blot analysis and immunocytochemistry, the authors evaluated the effect of α(2)M* on GFAP expression in the Müller cell line MIO-M1, which constitutively expresses LRP1. Intracellular signaling pathways activated by α(2)M* were examined by Western blot analysis. The effect of α(2)M* on GFAP expression in the mouse retina was examined by intravitreal microinjection of α(2)M* in mouse eyes. RESULTS These data demonstrate that α(2)M* induced GFAP expression in the MIO-M1 cell line, which was selectively blocked by RAP, an antagonist of LRP1 binding ligands. In addition, α(2)M* induced JAK/STAT pathway activation, determined by STAT3 phosphorylation (p-STAT3), which was also blocked by RAP. Finally, the authors showed that GFAP was expressed in the retinas of mice, preferentially in Müller cells at 3 and 6 days after a single intravitreal α(2)M* injection, whereas p-STAT3 staining increased at day 1 in both the ganglion cell layer and the inner nuclear layer. CONCLUSIONS These results demonstrate that α(2)M* induces GFAP expression in retinal Müller cells through LRP1, which could be mediated by JAK/STAT pathway activation.

Altered expression of matrix metalloproteinases and their tissue inhibitors as possible contributors to corneal droplet formation in climatic droplet keratopathy

Purpose:  Climatic droplet keratopathy (CDK) is an acquired corneal disease characterized by progressive scarring of the cornea. In several corneal diseases, matrix metalloproteinases (MMPs) are upregulated during the degradation of epithelial and stromal tissues. We investigated the levels, degree of activation and molecular forms of MMP‐2, MMP‐9, MMP‐8 and MMP‐13 and their tissue inhibitors TIMP‐1 and TIMP‐2 in tear fluid of patients with CDK.

A journey into the retina: Müller glia commanding survival and death

Müller glial cells (MGCs) are known to participate actively in retinal development and to contribute to homoeostasis through many intracellular mechanisms. As there are no homologous cells in other neuronal tissues, it is certain that retinal health depends on MGCs. These macroglial cells are located at the centre of the columnar subunit and have a great ability to interact with neurons, astrocytes, microglia and endothelial cells in order to modulate different events. Several investigations have focused their attention on the role of MGCs in diabetic retinopathy, a progressive pathology where several insults coexist. As expected, data suggest that MGCs display different responses according to the severity of the stimulus, and therefore trigger distinct events throughout the course of the disease. Here, we describe physiological functions of MGCs and their participation in inflammation, gliosis, synthesis and secretion of trophic and antioxidant factors in the diabetic retina. We invite the reader to consider the protective/deleterious role of MGCs in the early and late stages of the disease. In the light of the results, we open up the discussion around and ask the question: Is it possible that the modulation of a single cell type could improve or even re‐establish retinal function after an injury?