María C. Sánchez

Activated α2 macroglobulin induces matrix metalloproteinase 9 expression by low‐density lipoprotein receptor‐related protein 1 through MAPK‐ERK1/2 and NF‐κB activation in macrophage‐derived cell lines

Macrophages under certain stimuli induce matrix metalloproteinase 9 (MMP‐9) expression and protein secretion through the activation of MAPK‐ERK and NF‐κB signaling pathways. Previously, we demonstrated that activated α2‐macroglulin (α2M*) through the interaction with its receptor low‐density lipoprotein receptor‐related protein 1 (LRP1) induces macrophage proliferation mediated by the activation of MAPK‐ERK1/2. In the present work, we examined whether α2M*/LRP1interaction could induce the MMP‐9 production in J774 and Raw264.7 macrophage‐derived cell lines. It was shown that α2M* promoted MMP‐9 expression and protein secretion by LRP1 in both macrophage‐derived cell lines, which was mediated by the activation of MAPK‐ERK1/2 and NF‐κB. Both intracellular signaling pathways activated by α2M* were effectively blocked by calphostin‐C, suggesting involvement of PKC. In addition, we demonstrate that α2M* produced extracellular calcium influx via LRP1. However, when the intracellular calcium mobilization was inhibited by BAPTA‐AM, the α2M*‐induced MAPK‐ER1/2 activation was fully blocked in both macrophage cell lines. Finally, using specific pharmacological inhibitors for PKC, Mek1, and NF‐κB, it was shown that the α2M*‐induced MMP‐9 protein secretion was inhibited, indicating that the MMP production promoted by the α2M*/LRP1 interaction required the activation of both signaling pathways. These findings may prove useful in the understanding of the macrophage LRP1 role in the vascular wall during atherogenic plaque progression. J. Cell. Biochem. 111: 607–617, 2010.

Pregnancy-specific glycoprotein 1a activates dendritic cells to provide signals for Th17-, Th2-, and Treg-cell polarization

Because of their plasticity and central role in orchestrating immunity and tolerance, DCs can respond to pregnancy‐specific signals, thus promoting the appropriate immune response in order to support pregnancy. Here, we show that pregnancy‐specific glycoprotein (PSG1a), the major variant of PSG released into the circulation during pregnancy, targets DCs to differentiate into a subset with a unique phenotype and function. This semi‐mature phenotype is able to secrete IL‐6 and TGF‐β. PSG1a also affected the maturation of DCs, preventing the up‐regulation of some costimulatory molecules, and inducing the secretion of TGF‐β or IL‐10 and the expression of programmed death ligand 1 (PD‐L1) in response to TLR‐9 or CD40 ligation. In addition, PSG1a‐treated DCs promoted the enrichment of Th2‐type cytokines, IL‐17‐producing cells, and Treg cells from CD4+ T cells from DO11.10 Tg mice. Moreover, in vivo expression of PSG1a promoted the expansion of Ag‐specific CD4+CD25+Foxp3+ Treg cells and IL‐17‐, IL‐4‐, IL‐5‐, and IL‐10‐secreting cells able to protect against Listeria monocytogenes infection. Taken together, our data indicate that DCs can be targeted by PSG1a to generate the signals necessary to mount an appropriate, well‐balanced, and effective immune response able to protect against invading pathogens while at the same time being compatible with a successful pregnancy.

Insulin induces the low density lipoprotein receptor-related protein 1 (LRP1) degradation by the proteasomal system in J774 macrophage-derived cells

Low‐density lipoprotein receptor‐related protein 1 (LRP1) is an endocytic receptor, which binds and internalizes diverse ligands such as activated α2‐macroglobulin (α2M*). LRP1 promotes intracellular signaling, which downstream mediates cellular proliferation and migration of different types of cells, including macrophages. Unlike the LDL receptor, LRP1 expression is not sensitive to cellular cholesterol levels but appears to be responsive to insulin. It has been previously demonstrated that insulin increases the cell surface presentation of LRP1 in adipocytes and hepatocytes, which is mediated by the intracellular PI3K/Akt signaling activation. The LRP1 protein distribution is similar to other insulin‐regulated cell surface proteins, including transferring receptor (Tfr). However, in macrophages, the insulin effect on the LRP1 distribution and expression is not well characterized. Considering that macrophages play a central role in the pathogenesis of atherosclerosis, herein we evaluate the effect of insulin on the cellular expression of LRP1 in J774 macrophages‐derived cells using Western blot and immunofluorescence microscopy. Our data demonstrate that insulin induces a significant decrease in the LRP1 protein content, without changing the specific mRNA level of this receptor. Moreover, insulin specifically affected the protein expression of LRP1 but not Tfr. The insulin‐induced protein degradation of LRP1 in J774 cells was mediated by the activation of the PI3K/Akt pathway and proteasomal system by an enhanced ubiquitin–receptor conjugation. The decreased content of LRP1 induced by insulin affected the cellular internalization of α2M*. Thus, we propose that the protein degradation of LRP‐1 induced by insulin in macrophages could have important effects on the pathogenesis of atherosclerosis. J. Cell. Biochem. 106: 372–380, 2009.

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

Fasciola hepatica Kunitz Type Molecule Decreases Dendritic Cell Activation and Their Ability to Induce Inflammatory Responses

The complete repertoire of proteins with immunomodulatory activity in Fasciola hepatica (Fh) has not yet been fully described. Here, we demonstrated that Fh total extract (TE) reduced LPS-induced DC maturation, and the DC ability to induce allogeneic responses. After TE fractionating, a fraction lower than 10 kDa (F<10 kDa) was able to maintain the TE properties to modulate the DC pro- and anti-inflammatory cytokine production induced by LPS. In addition, TE or F<10 kDa treatment decreased the ability of immature DC to stimulate the allogeneic responses and induced a novo allogeneic CD4+CD25+Foxp3+ T cells. In contrast, treatment of DC with T/L or F<10 kDa plus LPS (F<10/L) induced a regulatory IL-27 dependent mechanism that diminished the proliferative and Th1 and Th17 allogeneic responses. Finally, we showed that a Kunitz type molecule (Fh-KTM), present in F<10 kDa, was responsible for suppressing pro-inflammatory cytokine production in LPS-activated DC, by printing tolerogenic features on DC that impaired their ability to induce inflammatory responses. These results suggest a modulatory role for this protein, which may be involved in the immune evasion mechanisms of the parasite.

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.

Structural evaluation of plasma α2-macroglobulin in acute pancreatitis

Abstract In this work we evaluate the proteolytic state of plasma α2-macroglobulin in acute pancreatitis. In addition, the plasma activity of matrix metalloproteinase-2 (MMP-2), MMP-9 and serine proteinases were analyzed. A total of 33 patients with acute pancreatitis were studied, of whom 16 were diagnosed as having mild and 17 as having severe acute pancreatitis. In the latter group, three patients progressed to multi-organ failure and died as a consequence of these complications. The proteolytic fragmentation of α2-macroglobulin was evaluated by Western blotting, whereas the plasma activity of MMP-2, MMP-9 and serine proteinases was evaluated by gelatin zymography. Enhanced fragmentation of α2-macroglobulin was detected in severe acute pancreatitis patients with multiple organ failure and lethal complications. In this same patient group, increased plasma activity of the active forms of MMP-2 and MMP-9, as well as serine proteinases, was apparent. In addition, we demonstrate that chymotrypsin-like proteinases could be the principal cause of α2-macroglobulin degradation in this group of patients. Our results indicate that secondary proteolysis of α2-macroglobulin promotes impaired control of extracellular proteolytic activity, leading to local and distant tissue injuries during severe acute pancreatitis. Finally, the structural evaluation of plasma α2-macroglobulin could be used as a prognostic marker of the severity of acute pancreatitis.

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.

Galectin-1 expression imprints a neurovascular phenotype in proliferative retinopathies and delineates responses to anti-VEGF

Neovascular retinopathies are leading causes of irreversible blindness. Although vascular endothelial growth factor (VEGF) inhibitors have been established as the mainstay of current treatment, clinical management of these diseases is still limited. As retinal impairment involves abnormal neovascularization and neuronal degeneration, we evaluated here the involvement of galectin-1 in vascular and non-vascular alterations associated with retinopathies, using the oxygen-induced retinopathy (OIR) model. Postnatal day 17 OIR mouse retinas showed the highest neovascular profile and exhibited neuro-glial injury as well as retinal functional loss, which persisted until P26 OIR. Concomitant to VEGF up-regulation, galectin-1 was highly expressed in P17 OIR retinas and it was mainly localized in neovascular tufts. In addition, OIR induced remodelling of cell surface glycophenotype leading to exposure of galectin-1-specific glycan epitopes. Whereas VEGF returned to baseline levels at P26, increased galectin-1 expression persisted until this time period. Remarkably, although anti-VEGF treatment in P17 OIR improved retinal vascularization, neither galectin-1 expression nor non-vascular and functional alterations were attenuated. However, this functional defect was partially prevented in galectin-1-deficient (Lgals1−/−) OIR mice, suggesting the importance of targeting both VEGF and galectin-1 as non-redundant independent pathways. Supporting the clinical relevance of these findings, we found increased levels of galectin-1 in aqueous humor from patients with proliferative diabetic retinopathy and neovascular glaucoma. Thus, using an OIR model and human samples, we identified a role for galectin-1 accompanying vascular and non-vascular retinal alterations in neovascular retinopathies.