Gustavo A. Chiabrando

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.

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

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.

Standardized flow cytometry assay for identification of human monocytic heterogeneity and LRP1 expression in monocyte subpopulations: Decreased expression of this receptor in nonclassical monocytes

In this article, we present a flow cytometry assay by which human blood monocyte subpopulations—classical (CD14++CD16−), intermediate (CD14++CD16+), and nonclassical (CD14+CD16++) monocytes—can be determined. Monocytic cells were selected from CD45+ leukocyte subsets by differential staining of the low‐density lipoprotein receptor‐related protein 1 (LRP1), which allows reducing the spill‐over of natural killer cells and granulocytes into the CD16+ monocyte gate. Percentages of monocyte subpopulations established by this procedure were significantly comparable with those obtained by a well‐standardized flow cytometry assay based on the HLA‐DR monocyte‐gating strategy. We also demonstrated that LRP1 is differentially expressed at cell surface of monocyte subpopulations, being significantly lower in nonclassical monocytes than in classical and intermediate monocytes. Cell surface expression of LRP1 accounts for only 20% of the total cellular content in each monocyte subpopulation. Finally, we established the within‐individual biological variation (bCV%) of circulating monocyte subpopulations in healthy donors, obtaining values of 21%, 20%, and 17% for nonclassical, intermediate, and classical monocytes, respectively. Similar values of bCV% for LRP1 measured in each monocyte subpopulation were also obtained, suggesting that its variability is mainly influenced by the intrinsic biological variation of circulating monocytes. Thus, we conclude that LRP1 can be used as a third pan‐monocytic marker together with CD14 and CD16 to properly identify monocyte subpopulations. The combined determination of monocyte subpopulations and LRP1 monocytic expression may be relevant for clinical studies of inflammatory processes, with special interest in atherosclerosis and cardiovascular disease.

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.

Decreased Expression of the Low-density Lipoprotein Receptor-related Protein-1 (LRP-1) in Rats with Prostate Cancer

The aim of this work was to evaluate by immunohistochemistry (IHC) the expression of both LRP-1 and urokinase-type plasminogen activator receptor (uPAR) at different developmental stages of rat prostate disease by using a prostate cancer model previously developed in our laboratory. We found that LRP-1 was weakly expressed in normal prostates and in rats with hyperplastic glands. The expression of this receptor increased and correlated with the degree of premalignant lesions (PIN I, II, and III). The IHC for uPAR in normal prostates and in premalignant lesions showed a score of immunostaining that correlated with the expression of LRP-1. On the other hand, in prostates with adenocarcinomas and undifferentiated carcinomas, LRP-1 was undetectable or weakly detected, whereas uPAR showed a significantly higher level of expression. Based on the IHC results in rat prostates with premalignant and malignant lesions and considering that LRP-1, by mediating the internalization of uPAR, is involved in the regulation of extracellular matrix remodeling and cell migration, we conclude that a decreased expression of LRP-1 could be involved with the increasing activation of plasminogen activators shown in cancers.

Differential reactivity of Agaricus bisporus lectin with human IgA subclasses in gel precipitation

The interaction between purified Agaricus bisporus lectin and several human proteins was studied using the Ouchterlony double diffusion and immunoelectrophoresis techniques. Only one precipitation line was observed with normal human serum, normal human colostrum, IgA1 myeloma serum, both serum monoclonal and secretory IgA1 and monoclonal IgD. No reaction was observed with monoclonal and secretory IgA2, IgG, IgM, alpha 2 macroglobulin or pregnancy-associated alpha 2 glycoprotein. These results were confirmed by hemagglutination inhibition assays when IgA1, IgA2 and IgD were tested. On the basis of this reactivity, ABL could be a useful tool for distinguishing and isolating human IgA subclasses.

Trypanosoma cruzi: cruzipain and membrane-bound cysteine proteinase isoform(s) interacts with human α2-macroglobulin and pregnancy zone protein

Plasmatic levels of pregnancy zone protein (PZP) increase in children with acute Chagas disease. PZP, as well as alpha2-macroglobulin (alpha2-M), are able to interact with Trypanosoma cruzi proteinases. The interaction of alpha2-M and PZP with cruzipain, the major cysteine proteinase of T. cruzi, was investigated. Several molecular changes on both alpha-M inhibitors under reaction with cruzipain were found. PAGE analysis showed: (i) formation of complexes of intermediate mobility and tetramerization of native alpha2-M and PZP, respectively; (ii) limited proteolysis of bait region in alpha2-M and PZP, and (iii) covalent binding of cruzipain to PZP and alpha2-M. Conformational and structural changes experimented by alpha-Ms correlate with modifications of the enzyme electrophoretic mobility and activity. Cruzipain-alpha-M complexes were also detected by gelatin SDS-PAGE and immunoblotting using polyclonal anti-cruzipain antibodies. Concomitantly, alpha2-M and PZP impaired the activity of cruzipain towards Bz-Pro-Phe-Arg-pNA substrate. In addition, alpha-Ms were able to form covalent complexes with membrane isoforms of cysteine proteinases cross-reacting with cruzipain. The present study suggests that both human alpha-macroglobulin inhibitors could prevent or minimize harmful action of cruzipain on hosts molecules and hypothetically regulate parasite functions controlled by cruzipain.