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.Fil: Jaldín Fincati, Javier Roberto. University of Toronto; Canadá. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba; ArgentinaFil: Actis Dato, Virginia. Universidad Nacional de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Díaz, Nicolás Maximiliano. Universidad Nacional de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Sánchez, María C.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba; ArgentinaFil: Barcelona, Pablo Federico. Universidad Nacional de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Chiabrando, Gustavo Alberto. Universidad Nacional de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentin

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-1β 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.Fil: Albertini, Ricardo Arturo. Hospital Privado Centro Medico de Cordoba; ArgentinaFil: Nicolas, Juan C.. Universidad Católica de Córdoba; ArgentinaFil: Actis Dato, Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Ferrer, Darío G.. Hospital Privado Centro Medico de Cordoba; ArgentinaFil: Tinti, María E.. Hospital Privado Centro Medico de Cordoba; ArgentinaFil: Capra, Raúl H.. Hospital Privado Centro Medico de Cordoba; ArgentinaFil: Chiabrando, Gustavo Alberto. Universidad Nacional de Córdoba; Argentina. Instituto Universitario de Ciencias Biomédicas de Córdoba; Argentin

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

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

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 (Gly-Cys) 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.The economic support to develop this project was received from Fundació MARATÓ TV3 with Grant 201521-10 (to V.L.-C.), FIS PI21/01523 (to V.L.-C.) from the Instituto de Salud Carlos III (ISCIII) and co-financed with ERDFs, and Fundación BBVA Ayudas a equipos de investigación 2019. This work was also funded by the Secretaría de Ciencia y Tecnología de la Universidad Nacional de Córdoba (SECyT-UNC) Grants PROYECTOS CONSOLIDAR 2018–2021 (to G.C.), Fondo para la Investigación Científica y Tecnológica (FONCyT), and Préstamo BID Proyecto de Investigación en Ciencia y Tecnología (PICT) Grants 2015-0807 and 2017-4497 (to G.C.). Support was received from the Albert Renold Travel Fellowship Programme 2019 from the European Foundation of the study of Diabetes (EFSD) and the Wood-Whelan fellowship Programme 2019 from the International Union of Biochemistry and Molecular Biology (IUBMB) to cover the stay of V.A.D. in Institute of Biomedical Research of Barcelona (IIBB)-Spanish National Research Council (CSIC) and Biomedical Research Institute Sant Pau (IIB Sant Pau). V.A.D. is a postdoctoral fellow of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Centro de Investigaciones en Bioquímica Clínica e Inmunología (CIBICI). A.B.-A. is a predoctoral fellow (FI19/00205) granted by the Programme _Contratos predoctorales de formación de investigación en salud_ from the Instituto de Salud Carlos III (ISCIII) and co-financed with ERDFs. Our group is part of CIBER Enfermedades Cardiovasculares (CIBERCV; CB16/11/00276 to J.M.G. and V.L.-C.) and CIBER Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM; CB07/08/0016 to J.C.E.-G.), projects run by the Instituto de Salud Carlos III. Our group also participates in Redes de investigación (Enfermedades Metabóloicas y Cáncer RED2018-102799-T), a project run by MINECO. The V.L.-C. group is recognized by Generalitat de Catalunya (2017 SGR 946). The authorsd thank Eva Prats and the staff from Centres Científics i Tecnològics de la Universitat de Barcelona (Campus Casanova) (CCiT/UB) for sample processing for electron microscopy. The IR-SANTPAU is a center of CERCA Programme/Generalitat de Catalunya

La inmunoterapia basada en LRP1 como estrategia eficaz para inhibir las alteraciones metabólicas en el sistema cardiovascular

Trabajo presentado en el Congreso SEC de la Salud Cardiovascular, celebrado en Mallorca (España), del 20 al 22 de octubre de 2022Background: 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

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-1β 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

Estudio de la relación funcional entre el tráfico intracelular de LRP1 y la respuesta celular a la insulina

Tesis (Doctora en Ciencias Químicas) - - Universidad Nacional de Córdoba. Facultad de Ciencias Químicas, 2019El LRP1 (low density lipoprotein receptor-related protein 1) es un receptor que se encuentra expresado en diferentes tipos celulares tales como células gliales de Müller y cardiomiocitos. Entre las diversas funciones de este receptor se destaca su capacidad de regular la expresión y actividad de diferentes proteínas en la membrana plasmática, como ser el caso del receptor de insulina (IR). Se ha demostrado que la deficiencia de LRP1 en neuronas y hepatocitos genera resistencia a la insulina. En este sentido, la insulina estimula el tráfico de LRP1 a la superficie celular en adipocitos, hepatocitos y células musculares, aunque se desconoce cómo es la regulación de este tráfico intracelular. Esta movilización de LRP1 hacia la membrana plasmática, a su vez, le permitiría regular la actividad del IR en respuesta a la hormona. Numerosos estudios relacionan la resistencia a la insulina con el incremento de subfracciones aterogénicas de lipoproteínas de baja densidad, como las modificadas por agregación (agLDL). El exceso de estas lipoproteínas es almacenado en diferentes tejidos, como músculo e hígado, contribuyendo con la resistencia a la insulina, aunque el mecanismo a través del cual esto ocurre no es del todo claro. El LRP1 es uno de los receptores involucrados en la captación de colesterol esterificado desde agLDL, lo que podría tener relación con alteración de la respuesta a la insulina. Teniendo en cuenta estos antecedentes, el objetivo del presente trabajo de tesis fue caracterizar la relación funcional entre el tráfico intracelular de LRP1 y la respuesta celular a la insulina. Dentro de este marco general se plantearon los siguientes objetivos específicos: i) Identificar las estructuras vesiculares de almacenamiento de LRP1, así como las proteínas de transporte vesicular y las vías de señalización intracelulares implicadas en el tráfico hacia la membrana plasmatica de este receptor por acción de insulina; y ii) Establecer la participación de LRP1 sobre la respuesta celular a insulina, y cómo este evento es alterado por la presencia de ligandos como las agLDL. 9 Como modelo experimental se utilizaron las líneas celulares: MIO-M1, células gliales de Müller de retinas humanas; y HL-1, cardiomiocitos de ratón. En cuanto al tráfico intracelular de LRP1 inducido por insulina en células MIO-M1, por microscopia confocal se evidenció un incremento significativo de la localización de LRP1 principalmente en endosomas tempranos [EEA1+] y en el compartimento de reciclado rápido [Rab4+] en presencia de insulina. Mediante ensayos de biotinilación de proteínas de superficie celular, se evidenció que insulina promueve la movilización de LRP1 hacia la membrana plasmática en estas células. Este tráfico de LRP1 hacia la membrana plasmática fue mediado por la activación de la vía de señalización intracelular IR/PI3K/Akt, evidenciado mediante ensayos de in cell Western blot. Por microscopia electrónica de trasmisión se pudo identificar vesículas intracelulares de ≈100 nm, que almacenan LRP1 junto con otras proteínas características de estas estructuras como sortilin y VAMP2. Finalmente, insulina promovió el tráfico de LRP1 a la superficie celular a través diferentes vías posibles de reciclado y/o exocitosis, entre ellas, la vía de reciclado rápido [Rab4+] y la vía exocítica mediada por Rab8A y Rab10, como se pudo observar mediante ensayos de biotinilacion de proteínas de superficie celular. En cuanto a la participación de LRP1 sobre la respuesta celular a insulina, y como este evento es afectado por las agLDL en células HL-1, se demostró que el tratamiento con agLDL durante 8 horas produjo una acumulación significativa de colesterol esterificado evidenciada por extracción lipídica y cromatografía en capa delgada. La captación de estas lipoproteínas modificadas fue mediada por LRP1, ya que este efecto fue bloqueado por GST-RAP. Mediante microscopia confocal se evidenció que la interacción entre las agLDL y LRP1 promovió la localización del receptor en compartimentos de degradación del tipo endosomas tardíos o lisosomas, los cuales resultaron ser disfuncionales, y afectó la localización de este receptor en regiones celulares ricas en caveolina por acción de insulina. Mediante ensayos de inmunoprecipitación, se evidenció la alteración de la asociación molecular entre LRP1 e IR. A continuación, mediante Western blot, ensayos de marcación de proteínas de superficie con biotina y microscopia confocal, encontramos que las agLDL afectaron la activación de la cascada de señalización 10 intracelular inducida por insulina lo que resultó en una reducción de la exocitosis hacia la membrana plasmática de GLUT4 y una menor captación de glucosa por acción de insulina en los cardiomiocitos expuestos a agLDL. En conclusión, la insulina regularía el tráfico de LRP1 hacia la membrana plasmática, principalmente desde vesículas intracelulares, a través de la activación de la cascada de señalización intracelular IR/PI3K/Akt, y en parte mediante una ruta de exocitosis regulada por insulina que involucraría la participación de Rab8A y Rab10. Este evento resulta en un aumento de la expresión de LRP1 en la superficie celular, lo cual tendría implicancias en la respuesta celular a la insulina. La presencia de un ligando específico de LRP1 como agLDL alteraría la distribución intracelular de este receptor, afectando la asociación molecular entre LRP1 e IR, la señalización intracelular inducida por insulina y la captación de glucosa extracelular en cardiomiocitos. Estos resultados en su conjunto presentan una nueva perspectiva en el estudio bioquímico y celular de la relación del tráfico intracelular de LRP1 y la respuesta celular a la insulina, lo que podría tener implicancias fisiopatológicas en los procesos de neovascularización y neurodegeneración retinal como así también en la alteración de la función cardíaca que se producen en situaciones de resistencia a la insulina.202

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 (LRP1—A 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

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.Fil: Actis Dato, Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Chiabrando, Gustavo Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentin

Metabolic Flexibility of the Heart: The Role of Fatty Acid Metabolism in Health, Heart Failure, and Cardiometabolic Diseases

This comprehensive review explores the critical role of fatty acid (FA) metabolism in cardiac diseases, particularly heart failure (HF), and the implications for therapeutic strategies. The heart's reliance on ATP, primarily sourced from mitochondrial oxidative metabolism, underscores the significance of metabolic flexibility, with fatty acid oxidation (FAO) being a dominant source. In HF, metabolic shifts occur with an altered FA uptake and FAO, impacting mitochondrial function and contributing to disease progression. Conditions like obesity and diabetes also lead to metabolic disturbances, resulting in cardiomyopathy marked by an over-reliance on FAO, mitochondrial dysfunction, and lipotoxicity. Therapeutic approaches targeting FA metabolism in cardiac diseases have evolved, focusing on inhibiting or stimulating FAO to optimize cardiac energetics. Strategies include using CPT1A inhibitors, using PPARα agonists, and enhancing mitochondrial biogenesis and function. However, the effectiveness varies, reflecting the complexity of metabolic remodeling in HF. Hence, treatment strategies should be individualized, considering that cardiac energy metabolism is intricate and tightly regulated. The therapeutic aim is to optimize overall metabolic function, recognizing the pivotal role of FAs and the need for further research to develop effective therapies, with promising new approaches targeting mitochondrial oxidative metabolism and FAO that improve cardiac function