Building momentum through networks: Bioimaging across the Americas.

In September 2023, the two largest bioimaging networks in the Americas, Latin America Bioimaging (LABI) and BioImaging North America (BINA), came together during a 1-week meeting in Mexico. This meeting provided opportunities for participants to interact closely with decision-makers from imaging core facilities across the Americas. The meeting was held in a hybrid format and attended in-person by imaging scientists from across the Americas, including Canada, the United States, Mexico, Colombia, Peru, Argentina, Chile, Brazil and Uruguay. The aims of the meeting were to discuss progress achieved over the past year, to foster networking and collaborative efforts among members of both communities, to bring together key members of the international imaging community to promote the exchange of experience and expertise, to engage with industry partners, and to establish future directions within each individual network, as well as common goals. This meeting report summarises the discussions exchanged, the achievements shared, and the goals set during the LABIxBINA2023: Bioimaging across the Americas meeting.

Transcriptional analysis reveals that the intracellular lipid accumulation impairs gene expression profiles involved in insulin response-associated cardiac functionality.

Cardiovascular disease (CVD) is a multisystemic and multicellular pathology that is generally associated with high levels of atherogenic lipoproteins in circulation. These lipoproteins tend to be retained and modified, for example, aggregated low-density lipoprotein (aggLDL), in the extracellular matrix of different tissues, such as the vascular wall and heart. The uptake of aggLDL generates a significant increase in cholesteryl ester (CE) in these tissues. We previously found that the accumulation of CE generates alterations in the insulin response in the heart. Although the insulin response is mainly associated with the uptake and metabolism of glucose, other studies have shown that insulin would fulfill functions in this tissue, such as regulating the calcium cycle and cardiac contractility. Here, we found that aggLDL induced-lipid accumulation altered the gene expression profile involved in processes essential for cardiac functionality, including insulin response and glucose uptake (Insr, Ins1, Pik3ip1, Slc2a4 gene expression), calcium cycle (Cacna1s and Gjc2 gene expression) and calcium-dependent cardiac contractility (Myh3), and cholesterol efflux (Abca1), in HL-1 cardiomyocytes. These observations were recapitulated using an in vivo model of hypercholesterolemic ApoE-KO mice. Altogether, these results may explain the deleterious effect of lipid accumulation in the myocardium, with important implications for lipid-overloaded associated CVD, including impaired insulin response, disrupted lipid metabolism, altered cardiac structure, and increased susceptibility to cardiovascular events.

Transcriptional analysis reveals that the intracellular lipid accumulation impairs gene expression profiles involved in insulin response-associated cardiac functionality.

Cardiovascular disease (CVD) is a multisystemic and multicellular pathology that is generally associated with high levels of atherogenic lipoproteins in circulation. These lipoproteins tend to be retained and modified, for example, aggregated low-density lipoprotein (aggLDL), in the extracellular matrix of different tissues, such as the vascular wall and heart. The uptake of aggLDL generates a significant increase in cholesteryl ester (CE) in these tissues. We previously found that the accumulation of CE generates alterations in the insulin response in the heart. Although the insulin response is mainly associated with the uptake and metabolism of glucose, other studies have shown that insulin would fulfill functions in this tissue, such as regulating the calcium cycle and cardiac contractility. Here, we found that aggLDL induced-lipid accumulation altered the gene expression profile involved in processes essential for cardiac functionality, including insulin response and glucose uptake ( Insr , Ins1 , Pik3ip1 , Slc2a4 gene expression), calcium cycle ( Cacna1s and Gjc2 gene expression) and calcium-dependent cardiac contractility ( Myh3 ), and cholesterol efflux ( Abca1 ), in HL-1 cardiomyocytes. These observations were recapitulated using an in vivo model of hypercholesterolemic ApoE-KO mice. Altogether, these results may explain the deleterious effect of lipid accumulation in the myocardium, with important implications for lipid-overloaded associated CVD.

Inhibitory Effects of LRP1-Based Immunotherapy on Cardiac Extracellular Matrix Biophysical Alterations Induced by Hypercholesterolemia.

The accumulation of lipids in cardiomyocytes contributes to cardiac dysfunction. The specific blockage of cardiomyocyte cholesteryl ester (CE) loading by antibodies (Abs) against the P3 sequence (Gly1127-Cys1140) of the LRP1 receptor improves cardiac insulin sensitivity. The impact of anti-P3 Abs on high-fat diet (HFD)-induced cardiac extracellular matrix (ECM) biophysical alterations was analyzed. Both IrP (without Abs) and P3-immunized rabbits (with Abs) were randomized into groups fed either HFD or a standard chow diet. Cardiac lipids, proteins, and carbohydrates were characterized by Fourier transform infrared spectroscopy in the attenuated total reflectance mode. The hydric organization and physical structure were determined by differential scanning calorimetry. HFD increased the levels of esterified lipids, collagen, and α-helical structures and upregulated fibrosis, bound water, and ECM plasticization in the heart. The inhibitory effect of anti-P3 Abs on cardiac CE accumulation was sufficient to reduce the collagen-filled extracellular space, the level of fibrosis, and the amount of bound water but did not counteract ECM plasticization in the heart of hypercholesterolemic rabbits.

Multitarget Activities of Müller Glial Cells and Low-Density Lipoprotein Receptor-Related Protein 1 in Proliferative Retinopathies.

Müller glial cells (MGCs), the main glial component of the retina, play an active role in retinal homeostasis during development and pathological processes. They strongly monitor retinal environment and, in response to retinal imbalance, activate neuroprotective mechanisms mainly characterized by the increase of glial fibrillary acidic protein (GFAP). Under these circumstances, if homeostasis is not reestablished, the retina can be severely injured and GFAP contributes to neuronal degeneration, as they occur in several proliferative retinopathies such as diabetic retinopathy, sickle cell retinopathy and retinopathy of prematurity. In addition, MGCs have an active participation in inflammatory responses releasing proinflammatory mediators and metalloproteinases to the extracellular space and vitreous cavity. MGCs are also involved in the retinal neovascularization and matrix extracellular remodeling during the proliferative stage of retinopathies. Interestingly, low-density lipoprotein receptor-related protein 1 (LRP1) and its ligand α2-macroglobulin (α2M) are highly expressed in MGCs and they have been established to participate in multiple cellular and molecular activities with relevance in retinopathies. However, the exact mechanism of regulation of retinal LRP1 in MGCs is still unclear. Thus, the active participation of MGCs and LRP1 in these diseases, strongly supports the potential interest of them for the design of novel therapeutic approaches. In this review, we discuss the role of LRP1 in the multiple MGCs activities involved in the development and progression of proliferative retinopathies, identifying opportunities in the field that beg further research in this topic area.Summary StatementMGCs and LRP1 are active players in injured retinas, participating in key features such as gliosis and neurotoxicity, neovascularization, inflammation, and glucose control homeostasis during the progression of ischemic diseases, such as proliferative retinopathies.

Decreased low-density lipoprotein receptor-related protein 1 expression in pro-inflammatory monocytes is associated with subclinical atherosclerosis.

Subclinical atherosclerosis (SCA) occurs in asymptomatic individuals. Blood peripheral monocytes are involved in the development of atherosclerosis. Circulating monocytes acquire pro-inflammatory profiles, and they are involved in the early stages of atherosclerosis development. Low-density lipoprotein Receptor-related Protein 1 (LRP1) is expressed in monocytes, mainly in classical and intermediate subsets. Although LRP1 is highly expressed in macrophages and vascular smooth muscle cells (VSMCs) in atherosclerotic plaque formation, its expression in circulating monocytes has not been studied in SCA. The aim of this study was to characterize the LRP1 expression level in circulating monocytes of individuals with SCA and compared with individuals with low (LR) and intermediate (IR) risk of cardiovascular diseases, both without evidence of atherosclerotic lesions in carotid and coronary arteries. LRP1 and additional markers (CD11b, CD11c, and CD36) at cell surface of monocytes were analyzed by flow cytometry assays, whereas LRP1 and pro-inflammatory factors gene expressions were measured in isolated monocytes by quantitative RT-PCRs. Both LRP1 protein and LRP1 mRNA were significantly reduced in monocytes in SCA and IR respect to LR. Conversely, CD36, CD11b, and CD11c monocytic markers showed no significant changes between the different study groups. Finally, increased gene expressions of TNF-α and IL-1ß were detected in monocytes of SCA, which were associated with decreased LRP1 expression at the cell surface in total monocytes. In summary, we propose that the decreased LRP1 expression at cell surface in total monocytes with pro-inflammatory profile is associated with the development of atherosclerosis in asymptomatic individuals.

Targeting cholesteryl ester accumulation in the heart improves cardiac insulin response.

BACKGROUND: Antibodies against the P3 sequence (Gly1127-Cys1140) of LRP1 (anti-P3 Abs) specifically block cholesteryl ester (CE) accumulation in vascular cells. LRP1 is a key regulator of insulin receptor (InsR) trafficking in different cell types. The link between CE accumulation and the insulin response are largely unknown. Here, the effects of P3 peptide immunization on the alterations induced by a high-fat diet (HFD) in cardiac insulin response were evaluated. METHODS: Irrelevant (IrP)- or P3 peptide-immunized rabbits were randomized into groups fed either HFD or normal chow. Cardiac lipid content was characterized by thin-layer chromatography, confocal microscopy, and electron microscopy. LRP1, InsR and glucose transporter type 4 (GLUT4) levels were determined in membranes and total lysates from rabbit heart. The interaction between InsR and LRP1 was analyzed by immunoprecipitation and confocal microscopy. Insulin signaling activity and glucose uptake were evaluated in HL-1 cells exposed to rabbit serum from the different groups. FINDINGS: HFD reduces cardiac InsR and GLUT4 membrane levels and the interactions between LRP1/InsR. Targeting the P3 sequence on LRP1 through anti-P3 Abs specifically reduces CE accumulation in the heart independently of changes in the circulating lipid profile. This restores InsR and GLUT4 levels in cardiac membranes as well as the LRP1/InsR interactions of HFD-fed rabbits. In addition, anti-P3 Abs restores the insulin signaling cascade and glucose uptake in HL-1 cells exposed to hypercholesterolemic rabbit serum. INTERPRETATION: LRP1-immunotargeting can block CE accumulation within the heart with specificity, selectivity, and efficacy, thereby improving the cardiac insulin response; this has important therapeutic implications for a wide range of cardiac diseases. FUNDING: Fundació MARATÓ TV3: grant 101521-10, Instiuto de Salud Carlos III (ISCIII) and ERDFPI18/01584, Fundación BBVA Ayudas a Equipos de Investigación 2019. SECyT-UNC grants PROYECTOS CONSOLIDAR 2018-2021; FONCyT, Préstamo BID PICT grant 2015-0807 and grant 2017-4497.

Activated Alpha-2 Macroglobulin Improves Insulin Response via LRP1 in Lipid-Loaded HL-1 Cardiomyocytes.

Activated alpha-2 Macroglobulin (α2M*) is specifically recognized by the cluster I/II of LRP1 (Low-density lipoprotein Receptor-related Protein-1). LRP1 is a scaffold protein for insulin receptor involved in the insulin-induced glucose transporter type 4 (GLUT4) translocation to plasma membrane and glucose uptake in different types of cells. Moreover, the cluster II of LRP1 plays a critical role in the internalization of atherogenic lipoproteins, such as aggregated Low-density Lipoproteins (aggLDL), promoting intracellular cholesteryl ester (CE) accumulation mainly in arterial intima and myocardium. The aggLDL uptake by LRP1 impairs GLUT4 traffic and the insulin response in cardiomyocytes. However, the link between CE accumulation, insulin action, and cardiac dysfunction are largely unknown. Here, we found that α2M* increased GLUT4 expression on cell surface by Rab4, Rab8A, and Rab10-mediated recycling through PI3K/Akt and MAPK/ERK signaling activation. Moreover, α2M* enhanced the insulin response increasing insulin-induced glucose uptake rate in the myocardium under normal conditions. On the other hand, α2M* blocked the intracellular CE accumulation, improved the insulin response and reduced cardiac damage in HL-1 cardiomyocytes exposed to aggLDL. In conclusion, α2M* by its agonist action on LRP1, counteracts the deleterious effects of aggLDL in cardiomyocytes, which may have therapeutic implications in cardiovascular diseases associated with hypercholesterolemia.

LRP1 mediates the IGF-1-induced GLUT1 expression on the cell surface and glucose uptake in Müller glial cells.

Insulin-like Growth Factor-1 (IGF-1) is involved in the normal development and survival of retinal cells. Low-density lipoprotein Receptor-related Protein-1 (LRP1) plays a key role on the regulation of several membrane proteins, such as the IGF-1 receptor (IGF-1R). In brain astrocytes, LRP1 interact with IGF-1R and the glucose transporter type 1 (GLUT1), regulating the glucose uptake in these cells. Although GLUT1 is expressed in retinal Müller Glial Cells (MGCs), its regulation is not clear yet. Here, we investigated whether IGF-1 modulates GLUT1 traffic to plasma membrane (PM) and glucose uptake, as well as the involvement of LRP1 in this process in the human Müller glial-derived cell line (MIO-M1). We found that IGF-1 produced GLUT1 translocation to the PM, in a time-dependent manner involving the intracellular signaling activation of MAPK/ERK and PI3K/Akt pathways, and generated a significant glucose uptake. Moreover, we found a molecular association between LRP1 and GLUT1, which was significantly reduced by IGF-1. Finally, cells treated with specific siRNA for LRP1 showed an impaired GLUT1 expression on PM and decreased glucose uptake induced by IGF-1. We conclude that IGF-1 regulates glucose homeostasis in MGCs involving the expression of LRP1.

Metabolic Syndrome Triggered by Fructose Diet Impairs Neuronal Function and Vascular Integrity in ApoE-KO Mouse Retinas: Implications of Autophagy Deficient Activation.

Metabolic syndrome is a disorder characterized by a constellation of clinical findings such as elevated blood glucose, hyperinsulinemia, dyslipidemia, hypertension, and obesity. A positive correlation has been found between metabolic syndrome or its components and retinopathy, mainly at microvascular level, in patients without a history of diabetes. Here, we extend the investigations beyond the vascular component analyzing functional changes as well as neuronal and glial response in retinas of Apolipoprotein E knockout (ApoE-KO) mice fed with 10% w/v fructose diet. Given that autophagy dysfunction is implicated in retinal diseases related to hyperglycemia and dyslipidemia, the activation of this pathway was also analyzed. Two months of fructose intake triggered metabolic derangements in ApoE-KO mice characterized by dyslipidemia, hyperglycemia and hyperinsulinemia. An increased number of TUNEL positive cells, in addition to the ganglion cell layer, was observed in the inner nuclear layer in retina. Vascular permeability, evidenced by albumin-Evans blue leakage and extravasation of albumin was also detected. Furthermore, a significant decrease of the glial fibrillary acidic protein expression was confirmed by Western blot analysis. Absence of both Müller cell gliosis and pro-angiogenic response was also demonstrated. Finally, retinas of ApoE-KO FD mice showed defective autophagy activation as judged by LC3B mRNA and p62 protein levels correlating with the increased cell death. These results demonstrated that FD induced in ApoE-KO mice biochemical alterations compatible with metabolic syndrome associated with neuronal impairment and mild vascular alterations in the retina.

Nitro-oleic acid, a ligand of CD36, reduces cholesterol accumulation by modulating oxidized-LDL uptake and cholesterol efflux in RAW264.7 macrophages.

Macrophages play a pivotal role in the early stages of atherosclerosis development; they excessively accumulate cholesterol in the cytosol in response to modified Low Density Lipoprotein (mLDL). The mLDL are incorporated through scavenger receptors. CD36 is a high-affinity cell surface scavenger receptor that facilitates the binding and uptake of long-chain fatty acids and mLDL into the cell. Numerous structurally diverse ligands can initiate signaling responses through CD36 to regulate cell metabolism, migration, and angiogenesis. Nitro-fatty acids are endogenous electrophilic lipid mediators that react with and modulate the function of multiple enzymes and transcriptional regulatory proteins. These actions induce the expression of several anti-inflammatory and cytoprotective genes and limit pathologic responses in experimental models of atherosclerosis, cardiac ischemia/reperfusion, and inflammatory diseases. Pharmacological and genetic approaches were used to explore the actions of nitro-oleic acid (NO2-OA) on macrophage lipid metabolism. Pure synthetic NO2-OA dose-dependently increased CD36 expression in RAW264.7 macrophages and this up-regulation was abrogated in BMDM from Nrf2-KO mice. Ligand binding analysis revealed that NO2-OA specifically interacts with CD36, thus limiting the binding and uptake of mLDL. Docking analysis shows that NO2-OA establishes a low binding energy interaction with the alpha helix containing Lys164 in CD36. NO2-OA also restored autophagy flux in mLDL-loaded macrophages, thus reversing cholesterol deposition within the cell. In aggregate, these results indicate that NO2-OA reduces cholesterol uptake by binding to CD36 and increases cholesterol efflux by restoring autophagy.

LRP1-Mediated AggLDL Endocytosis Promotes Cholesteryl Ester Accumulation and Impairs Insulin Response in HL-1 Cells.

Abstract: The cardiovascular disease (CVD) frequently developed during metabolic syndrome and type-2 diabetes mellitus is associated with increased levels of aggregation-prone small LDL particles. Aggregated LDL (aggLDL) internalization is mediated by low-density lipoprotein receptor-related protein-1 (LRP1) promoting intracellular cholesteryl ester (CE) accumulation. Additionally, LRP1 plays a key function in the regulation of insulin receptor (IR) and glucose transporter type 4 (GLUT4) activities. Nevertheless, the link between LRP1, CE accumulation, and insulin response has not been previously studied in cardiomyocytes. We aimed to identify mechanisms through which aggLDL, by its interaction with LRP1, produce CE accumulation and affects the insulin-induced intracellular signaling and GLUT4 trafficking in HL-1 cells. We demonstrated that LRP1 mediates the endocytosis of aggLDL and promotes CE accumulation in these cells. Moreover, aggLDL reduced the molecular association between IR and LRP1 and impaired insulin-induced intracellular signaling activation. Finally, aggLDL affected GLUT4 translocation to the plasma membrane and the 2-NBDG uptake in insulin-stimulated cells. We conclude that LRP1 is a key regulator of the insulin response, which can be altered by CE accumulation through LRP1-mediated aggLDL endocytosis.

Effects of chlorogenic acid on thermal stress tolerance in C. elegans via HIF-1, HSF-1 and autophagy.

BACKGROUND: Chlorogenic acid (CGA) is a polyphenol widely distributed in plants and plant-derived food with antioxidant and protective activities against cell stress. Caenorhabditis elegans is a model organism particularly useful for understanding the molecular and biochemical mechanisms associated with aging and stress in mammals. In C. elegans, CGA was shown to improve resistance to thermal, while the underlying mechanisms that lead to this effect require further understanding. PURPOSE: The present study was conducted to investigate the underlying molecular mechanisms behind CGA response conferring thermotolerance to C. elegans. METHODS AND RESULTS: Signaling pathways that could be involved in the CGA-induced thermotolerance were evaluated in C. elegans strains with loss-of-function mutation. CGA-induced thermotolerance required hypoxia-inducible factor HIF-1 but no insulin pathway. CGA exposition (1.4 µM CGA for 18 h) before thermal stress treatment increased HIF-1 levels and activity. HIF-1 activation could be partly attributed to an increase in radical oxygen species and a decrease in superoxide dismutase activity. In addition, CGA exposition before thermal stress also increased autophagy just as hormetic heat condition (HHC), worms incubated at 36 °C for 1 h. RNAi experiments evidenced that autophagy was increased by CGA via HIF-1, heat-shock transcription factor HSF-1 and heat-shock protein HSP-16 and HSP-70. In contrast, autophagy induced by HHC only required HSF-1 and HSP-70. Moreover, suppression of autophagy induction showed the significance of this process for adapting C. elegans to cope with thermal stress. CONCLUSION: This study demonstrates that CGA-induced thermotolerance in C. elegans is mediated by HIF-1 and downstream, by HSF-1, HSPs and autophagy resembling HHC.

Activated α2-Macroglobulin Regulates LRP1 Levels at the Plasma Membrane through the Activation of a Rab10-dependent Exocytic Pathway in Retinal Müller Glial Cells.

Activated α2-macroglobulin (α2M*) and its receptor, low-density lipoprotein receptor-related protein 1 (LRP1), have been linked to proliferative retinal diseases. In Müller glial cells (MGCs), the α2M*/LRP1 interaction induces cell signaling, cell migration, and extracellular matrix remodeling, processes closely associated with proliferative disorders. However, the mechanism whereby α2M* and LRP1 participate in the aforementioned pathologies remains incompletely elucidated. Here, we investigate whether α2M* regulates both the intracellular distribution and sorting of LRP1 to the plasma membrane (PM) and how this regulation is involved in the cell migration of MGCs. Using a human Müller glial-derived cell line, MIO-M1, we demonstrate that the α2M*/LRP1 complex is internalized and rapidly reaches early endosomes. Afterward, α2M* is routed to degradative compartments, while LRP1 is accumulated at the PM through a Rab10-dependent exocytic pathway regulated by PI3K/Akt. Interestingly, Rab10 knockdown reduces both LRP1 accumulation at the PM and cell migration of MIO-M1 cells induced by α2M*. Given the importance of MGCs in the maintenance of retinal homeostasis, unravelling this molecular mechanism can potentially provide new therapeutic targets for the treatment of proliferative retinopathies.

Effect of Autophagy Modulators on Vascular, Glial, and Neuronal Alterations in the Oxygen-Induced Retinopathy Mouse Model.

Hypoxia is one of the main insults in proliferative retinopathies, leading to neovascularization and neurodegeneration. To maintain homeostasis, neurons require efficient degradation and recycling systems. Autophagy participates in retinal cell death, but it is also a cell survival mechanism. Here, we analyzed the role of autophagy at the three characteristic time periods in the oxygen-induced retinopathy (OIR) mouse model and determined if its modulation can improve vascular and non-vascular alterations. Experiments were performed with chloroquine (CQ) in order to monitor autophagosome accumulation by lysosomal blockade. Post natal day (P)17 OIR mouse retinas showed a significant increase in autophagy flux. In particular, an intense LC3B and p62 staining was observed in inner layers of the retina, mainly proliferating endothelial cells. After a single intraocular injection of Rapamycin at P12 OIR, a decreased neovascular area and vascular endothelial growth factor (VEGF) protein expression were observed at P17 OIR. In addition, whereas the increased expression of glial fibrillary acidic protein (GFAP) was reversed at P26 OIR, the functional alterations persisted. Using a similar therapeutic schedule, we analyzed the effect of anti-VEGF therapy on autophagy flux. Like Rapamycin, VEGF inhibitor treatment not only reduced the amount of neovascular tufts, but also activated autophagy flux at P17 OIR, mainly in ganglion cell layer and inner nuclear layer. Finally, the effects of the disruption of autophagy by Spautin-1, were evaluated at vascular, glial, and neuronal levels. After a single dose of Spautin-1, Western blot analysis showed a significant decrease in LC3B II and p62 protein expression at P13 OIR, returning both autophagy markers to OIR control levels at P17. In addition, neither gliosis nor functional alterations were attenuated. In line with these results, TUNEL staining showed a slight increase in the number of positive cells in the outer nuclear layer at P17 OIR. Overall, our results demonstrate that all treatments of induction or inhibition of the autophagic flux reduced neovascular area but were unable to completely reverse the neuronal damage. Besides, compared to current treatments, rapamycin provides a more promising therapeutic strategy as it reduces both neovascular tufts and persistent gliosis.

Hemin induces autophagy in a leukemic erythroblast cell line through the LRP1 receptor.

Hemin is an erythropoietic inductor capable of inducing autophagy in erythroid-like cell lines. Low-density lipoprotein receptor-related protein 1 (LRP1) is a transmembrane receptor involved in a wide range of cellular processes, such as proliferation, differentiation, and metabolism. Our aim was to evaluate whether LRP1 is responsible for hemin activity in K562 cells, with the results demonstrating a three-fold increase in LRP1 gene expression levels (P-values <0.001) when assessed by quantitative real-time RT-PCR (qRT-PCR). Moreover, a 70% higher protein amount was observed compared with control condition (P-values <0.01) by Western blot (WB). Time kinetic assays demonstrated a peak in light chain 3 (LC3) II (LC3II) levels after 8 h of hemin stimulation and the localization of LRP1 in the autophagosome structures. Silencing LRP1 by siRNA decreased drastically the hemin-induced autophagy activity by almost 80% compared with control cells (P-values <0.01). Confocal localization and biochemical analysis indicated a significant redistribution of LRP1 from early endosomes and recycling compartments to late endosomes and autophagolysosomes, where the receptor is degraded. We conclude that LRP1 is responsible for hemin-induced autophagy activity in the erythroblastic cell line and that hemin-LRP1 complex activation promotes a self-regulation of the receptor. Our results suggest that hemin, via the LRP1 receptor, favors erythroid maturation by inducing an autophagic response, making it a possible therapeutic candidate to help in the treatment of hematological disorders.

The Role of Low-Density Lipoprotein Receptor-Related Protein 1 in Lipid Metabolism, Glucose Homeostasis and Inflammation.

Metabolic syndrome (MetS) is a highly prevalent disorder which can be used to identify individuals with a higher risk for cardiovascular disease and type 2 diabetes. This metabolic syndrome is characterized by a combination of physiological, metabolic, and molecular alterations such as insulin resistance, dyslipidemia, and central obesity. The low-density lipoprotein receptor-related protein 1 (LRP1­A member of the LDL receptor family) is an endocytic and signaling receptor that is expressed in several tissues. It is involved in the clearance of chylomicron remnants from circulation, and has been demonstrated to play a key role in the lipid metabolism at the hepatic level. Recent studies have shown that LRP1 is involved in insulin receptor (IR) trafficking and intracellular signaling activity, which have an impact on the regulation of glucose homeostasis in adipocytes, muscle cells, and brain. In addition, LRP1 has the potential to inhibit or sustain inflammation in macrophages, depending on its cellular expression, as well as the presence of particular types of ligands in the extracellular microenvironment. In this review, we summarize existing perspectives and the latest innovations concerning the role of tissue-specific LRP1 in lipoprotein and glucose metabolism, and examine its ability to mediate inflammatory processes related to MetS and atherosclerosis.

Insulin-induced exocytosis regulates the cell surface level of low-density lipoprotein-related protein-1 in Müller Glial cells.

Low-density lipoprotein (LDL) receptor-related protein-1 (LRP1) is expressed in retinal Müller glial cells (MGCs) and regulates intracellular translocation to the plasma membrane (PM) of the membrane proteins involved in cellular motility and activity. Different functions of MGCs may be influenced by insulin, including the removal of extracellular glutamate in the retina. In the present work, we investigated whether insulin promotes LRP1 translocation to the PM in the Müller glial-derived cell line MIO-M1 (human retinal Müller glial cell-derived cell line). We demonstrated that LRP1 is stored in small vesicles containing an approximate size of 100 nm (mean diameter range of 100-120 nm), which were positive for sortilin and VAMP2, and also incorporated GLUT4 when it was transiently transfected. Next, we observed that LRP1 translocation to the PM was promoted by insulin-regulated exocytosis through intracellular activation of the IR/PI3K/Akt axis and Rab-GTPase proteins such as Rab8A and Rab10. In addition, these Rab-GTPases regulated both the constitutive and insulin-induced LRP1 translocation to the PM. Finally, we found that dominant-negative Rab8A and Rab10 mutants impaired insulin-induced intracellular signaling of the IR/PI3K/Akt axis, suggesting that these GTPase proteins as well as the LRP1 level at the cell surface are involved in insulin-induced IR activation.

IGF-1R Regulates the Extracellular Level of Active MMP-2, Pathological Neovascularization, and Functionality in Retinas of OIR Mouse Model.

In ischemic proliferative diseases such as retinopathies, persistent hypoxia leads to the release of numerous neovascular factors that participate in the formation of abnormal vessels and eventually cause blindness. The upregulation and activation of metalloproteinases (MMP-2 and MMP-9) represent a final common pathway in this process. Although many regulators of the neovascular process have been identified, the complete role of the insulin-like growth factor 1 (IGF-1) and its receptor (IGF-1R) appears to be significantly more complex. In this study, we used an oxygen-induced retinopathy (OIR) mouse model as well as an in vitro model of hypoxia to study the role of MMP-2 derived from Müller glial cells (MGCs) and its relation with the IGF-1/IGF-1R system. We demonstrated that MMP-2 protein expression increased in P17 OIR mice, which coincided with the active phase of the neovascular process. Also, glutamine synthetase (GS)-positive cells were also positive for MMP-2, whereas IGF-1R was expressed by GFAP-positive cells, indicating that both proteins were expressed in MGCs. In addition, in the OIR model a single intravitreal injection of the IGF-1R blocking antibody (αIR3) administered at P12 effectively prevented pathologic neovascularization, accelerated physiological revascularization, and improved retinal functionality at P17. Finally, in MGC supernatants, the blocking antibody abolished the IGF-1 effect on active MMP-2 under normoxic and hypoxic conditions without affecting the extracellular levels of pro-MMP-2. These results demonstrate, for the first time, that the IGF-1/IGF-1R system regulates active MMP-2 levels in MGCs, thus contributing to MEC remodeling during the retinal neovascular process.