Cholesterol trafficking, lysosomal function, and atherosclerosis.

Despite years of study and major research advances over the past 50 years, atherosclerotic diseases continue to rank as the leading global cause of death. Accumulation of cholesterol within the vascular wall remains the main problem and represents one of the early steps in the development of atherosclerotic lesions. There is a complex relationship between vesicular cholesterol transport and atherosclerosis, and abnormalities in cholesterol trafficking can contribute to the development and progression of the lesions. The dysregulation of vesicular cholesterol transport and lysosomal function fosters the buildup of cholesterol within various intracytoplasmic compartments, including lysosomes and lipid droplets. This, in turn, promotes the hallmark formation of foam cells, a defining feature of early atherosclerosis. Multiple cellular processes, encompassing endocytosis, exocytosis, intracellular trafficking, and autophagy, play crucial roles in influencing foam cell formation and atherosclerotic plaque stability. In this review, we highlight recent advances in the understanding of the intricate mechanisms of vesicular cholesterol transport and its relationship with atherosclerosis and discuss the importance of understanding these mechanisms in developing strategies to prevent or treat this prevalent cardiovascular disease.

Wnt5a Promotes Lysosomal Cholesterol Egress and Protects Against Atherosclerosis.

BACKGROUND: Impairment of cellular cholesterol trafficking is at the heart of atherosclerotic lesions formation. This involves egress of cholesterol from the lysosomes and 2 lysosomal proteins, the NPC1 (Niemann-Pick C1) and NPC2 that promotes cholesterol trafficking. However, movement of cholesterol out the lysosome and how disrupted cholesterol trafficking leads to atherosclerosis is unclear. As the Wnt ligand, Wnt5a inhibits the intracellular accumulation of cholesterol in multiple cell types, we tested whether Wnt5a interacts with the lysosomal cholesterol export machinery and studied its role in atherosclerotic lesions formation. METHODS: We generated mice deleted for the Wnt5a gene in vascular smooth muscle cells. To establish whether Wnt5a also protects against cholesterol accumulation in human vascular smooth muscle cells, we used a CRISPR/Cas9 guided nuclease approach to generate human vascular smooth muscle cells knockout for Wnt5a. RESULTS: We show that Wnt5a is a crucial component of the lysosomal cholesterol export machinery. By increasing lysosomal acid lipase expression, decreasing metabolic signaling by the mTORC1 (mechanistic target of rapamycin complex 1) kinase, and through binding to NPC1 and NPC2, Wnt5a senses changes in dietary cholesterol supply and promotes lysosomal cholesterol egress to the endoplasmic reticulum. Consequently, loss of Wnt5a decoupled mTORC1 from variations in lysosomal sterol levels, disrupted lysosomal function, decreased cholesterol content in the endoplasmic reticulum, and promoted atherosclerosis. CONCLUSIONS: These results reveal an unexpected function of the Wnt5a pathway as essential for maintaining cholesterol homeostasis in vivo.

atherosclerosis: gone with the Wnt?

Atherosclerosis, a pathology affecting large and medium-sized arteries, is the major cause of cardiovascular morbidity/mortality in industrialized countries. During atherosclerosis, cells accumulate large amounts of cholesterol through the uptake of modified low-density lipoprotein particles to form foam cells. This accumulation forms the basis for the development of the disease and for a large spectrum of other diseases in various organs. Massive research efforts have yielded valuable information about the underlying molecular mechanisms of atherosclerosis. In particular, newer discoveries on the early stage of lesion formation, cholesterol accumulation, reverse cholesterol transport, and local inflammation in the vascular wall have opened unanticipated horizons of understanding and raised novel questions and therapeutic opportunities. In this review, we focus on Wnt signaling, which has received little attention so far, yet affects lysosomal function and signalling pathways that limit cholesterol accumulation. This occurs in different tissues and cell types, including smooth muscle cells, endothelial cells and macrophages in the arterial wall, and thus profoundly impacts on atherosclerotic disease development and progression.

Author Correction: Loss of the adaptor protein ShcA in endothelial cells protects against monocyte macrophage adhesion, LDL-oxydation, and atherosclerotic lesion formation.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Loss of the adaptor protein ShcA in endothelial cells protects against monocyte macrophage adhesion, LDL-oxydation, and atherosclerotic lesion formation.

ShcA is an adaptor protein that binds to the cytoplasmic tail of receptor tyrosine kinases and of the Low Density Lipoprotein-related receptor 1 (LRP1), a trans-membrane receptor that protects against atherosclerosis. Here, we examined the role of endothelial ShcA in atherosclerotic lesion formation. We found that atherosclerosis progression was markedly attenuated in mice deleted for ShcA in endothelial cells, that macrophage content was reduced at the sites of lesions, and that adhesion molecules such as the intercellular adhesion molecule-1 (ICAM-1) were severely reduced. Our data indicate that transcriptional regulation of ShcA by the zinc-finger E-box-binding homeobox 1 (ZEB1) and the Hippo pathway effector YAP, promotes ICAM-1 expression independently of p-NF-κB, the primary driver of adhesion molecules expressions. In addition, ShcA suppresses endothelial Akt and nitric oxide synthase (eNOS) expressions. Thus, through down regulation of eNOS and ZEB1-mediated ICAM-1 up regulation, endothelial ShcA promotes monocyte-macrophage adhesion and atherosclerotic lesion formation. Reducing ShcA expression in endothelial cells may represent an obvious therapeutic approach to prevent atherosclerosis.

Convergent Signaling Pathways Controlled by LRP1 (Receptor-related Protein 1) Cytoplasmic and Extracellular Domains Limit Cellular Cholesterol Accumulation.

The low density lipoprotein receptor-related protein 1 (LRP1) is a ubiquitously expressed cell surface receptor that protects from intracellular cholesterol accumulation. However, the underlying mechanisms are unknown. Here we show that the extracellular (α) chain of LRP1 mediates TGFß-induced enhancement of Wnt5a, which limits intracellular cholesterol accumulation by inhibiting cholesterol biosynthesis and by promoting cholesterol export. Moreover, we demonstrate that the cytoplasmic (ß) chain of LRP1 suffices to limit cholesterol accumulation in LRP1(-/-) cells. Through binding of Erk2 to the second of its carboxyl-terminal NPXY motifs, LRP1 ß-chain positively regulates the expression of ATP binding cassette transporter A1 (ABCA1) and of neutral cholesterol ester hydrolase (NCEH1). These results highlight the unexpected functions of LRP1 and the canonical Wnt5a pathway and new therapeutic potential in cholesterol-associated disorders including cardiovascular diseases.

The Src homology and collagen A (ShcA) adaptor protein is required for the spatial organization of the costamere/Z-disk network during heart development.

Src homology and collagen A (ShcA) is an adaptor protein that binds to tyrosine kinase receptors. Its germ line deletion is embryonic lethal with abnormal cardiovascular system formation, and its role in cardiovascular development is unknown. To investigate its functional role in cardiovascular development in mice, ShcA was deleted in cardiomyocytes and vascular smooth muscle cells by crossing ShcA flox mice with SM22a-Cre transgenic mice. Conditional mutant mice developed signs of severe dilated cardiomyopathy, myocardial infarctions, and premature death. No evidence of a vascular contribution to the phenotype was observed. Histological analysis of the heart revealed aberrant sarcomeric Z-disk and M-band structures, and misalignments of T-tubules with Z-disks. We find that not only the ErbB3/Neuregulin signaling pathway but also the baroreceptor reflex response, which have been functionally associated, are altered in the mutant mice. We further demonstrate that ShcA interacts with Caveolin-1 and the costameric protein plasma membrane Ca(2+)/calmodulin-dependent ATPase (PMCA), and that its deletion leads to abnormal dystrophin signaling. Collectively, these results demonstrate that ShcA interacts with crucial proteins and pathways that link Z-disk and costamere.

The nuclear hormone receptor PPARγ counteracts vascular calcification by inhibiting Wnt5a signalling in vascular smooth muscle cells.

Vascular calcification is a hallmark of advanced atherosclerosis. Here we show that deletion of the nuclear receptor PPARγ in vascular smooth muscle cells of low density lipoprotein receptor (LDLr)-deficient mice fed an atherogenic diet high in cholesterol, accelerates vascular calcification with chondrogenic metaplasia within the lesions. Vascular calcification in the absence of PPARγ requires expression of the transmembrane receptor LDLr-related protein-1 in vascular smooth muscle cells. LDLr-related protein-1 promotes a previously unknown Wnt5a-dependent prochondrogenic pathway. We show that PPARγ protects against vascular calcification by inducing the expression of secreted frizzled-related protein-2, which functions as a Wnt5a antagonist. Targeting this signalling pathway may have clinical implications in the context of common complications of atherosclerosis, including coronary artery calcification and valvular sclerosis.

Differential signaling by adaptor molecules LRP1 and ShcA regulates adipogenesis by the insulin-like growth factor-1 receptor.

The low density lipoprotein receptor-related protein (LRP1) is a transmembrane receptor that integrates multiple signaling pathways. Its cytoplasmic domain serves as docking sites for several adaptor proteins such as the Src homology 2/α-collagen (ShcA), which also binds to several tyrosine kinase receptors such as the insulin-like growth factor 1 (IGF-1) receptor. However, the physiological significance of the physical interaction between LRP1 and ShcA, and whether this interaction modifies tyrosine kinase receptor signaling, are still unknown. Here we report that LRP1 forms a complex with the IGF-1 receptor, and that LRP1 is required for ShcA to become sensitive to IGF-1 stimulation. Upon IGF-1 treatment, ShcA is tyrosine phosphorylated and translocates to the plasma membrane only in the presence of LRP1. This leads to the recruitment of the growth factor receptor-bound protein 2 (Grb2) to ShcA, and activation of the Ras/MAP kinase pathway. Conversely, in the absence of ShcA, IGF-1 signaling bifurcates toward the Akt/mammalian target of rapamycin pathway and accelerates adipocyte differentiation when cells are stimulated for adipogenesis. These results establish the LRP1-ShcA complex as an essential component in the IGF-1-regulated pathway for MAP kinase and Akt/mammalian target of rapamycin activation, and may help to understand the IGF-1 signaling shift from clonal expansion to growth-arrested cells and differentiation during adipogenesis.

LRP1 controls intracellular cholesterol storage and fatty acid synthesis through modulation of Wnt signaling.

The low-density lipoprotein receptor-related protein LRP1 is a cell surface receptor with functions in diverse physiological pathways, including lipid metabolism. Here we show that LRP1-deficient fibroblasts accumulate high levels of intracellular cholesterol and cholesteryl-ester when stimulated for adipocyte differentiation. We demonstrate that LRP1 stimulates a canonical Wnt5a signaling pathway that prevents cholesterol accumulation. Moreover, we show that LRP1 is required for lipolysis and stimulates fatty acid synthesis independently of the noradrenergic pathway, through inhibition of GSK3beta and its previously unknown target acetyl-CoA carboxylase (ACC). As a result of ACC inhibition, mature LRP1-deficient adipocytes of adult mice are hypotrophic, and lower uptake of fatty acids into adipose tissue leads to their redistribution to the liver. These results establish LRP1 as a novel integrator of adipogenic differentiation and fat storage signals.

LRP1 functions as an atheroprotective integrator of TGFbeta and PDFG signals in the vascular wall: implications for Marfan syndrome.

BACKGROUND: The multifunctional receptor LRP1 controls expression, activity and trafficking of the PDGF receptor-beta in vascular smooth muscle cells (VSMC). LRP1 is also a receptor for TGFbeta1 and is required for TGFbeta mediated inhibition of cell proliferation. METHODS AND PRINCIPAL FINDINGS: We show that loss of LRP1 in VSMC (smLRP(-)) in vivo results in a Marfan-like syndrome with nuclear accumulation of phosphorylated Smad2/3, disruption of elastic layers, tortuous aorta, and increased expression of the TGFbeta target genes thrombospondin-1 (TSP1) and PDGFRbeta in the vascular wall. Treatment of smLRP1(-) animals with the PPARgamma agonist rosiglitazone abolished nuclear pSmad accumulation, reversed the Marfan-like phenotype, and markedly reduced smooth muscle proliferation, fibrosis and atherosclerosis independent of plasma cholesterol levels. CONCLUSIONS AND SIGNIFICANCE: Our findings are consistent with an activation of TGFbeta signals in the LRP1-deficient vascular wall. LRP1 may function as an integrator of proliferative and anti-proliferative signals that control physiological mechanisms common to the pathogenesis of Marfan syndrome and atherosclerosis, and this is essential for maintaining vascular wall integrity.

The LDL receptor-related protein (LRP) family: an old family of proteins with new physiological functions.

The low-density lipoprotein (LDL) receptor is the founding member of a family of seven structurally closely related transmembrane proteins (LRP1, LRP1b, megalin/LRP2, LDL receptor, very low-density lipoprotein receptor, MEGF7/LRP4, LRP8/apolipoprotein E receptor2). These proteins participate in a wide range of physiological processes, including the regulation of lipid metabolism, protection against atherosclerosis, neurodevelopment, and transport of nutrients and vitamins. While currently available data suggest that the role of the LDL receptor is limited to the regulation of cholesterol homeostasis by receptor-mediated endocytosis of lipoprotein particles, there is growing experimental evidence that the other members of the gene family have additional physiological functions as signal transducers. In this review, we focus on the latest discovered functions of two major members of this family, LRP1 and megalin/LRP2, and on the newly elucidated physiological role of a third member of the family, MEGF7/LRP4, which can also function as a modulator of diverse signaling pathways during development.

Promotion of leukocyte adhesion by a novel interaction between vitronectin and the beta2 integrin Mac-1 (alphaMbeta2, CD11b/CD18).

OBJECTIVE: The leukocyte integrin Mac-1 (alphaMbeta2, CD11b/CD18) binds a number of ligands and counter-receptors and thereby is a major determinant in regulation of leukocyte adhesion and extravasation. Vitronectin (VN) is an adhesion-promoting factor that is abundantly present as matrix molecule in vascular diseases such as atherosclerosis. Until now, only an indirect interaction between Mac-1 and VN via the urokinase receptor (urokinase plasminogen activator receptor) was known. We now propose that Mac-1 and VN can directly interact with each other. METHODS AND RESULTS: In an in vitro system with purified components, Mac-1 specifically bound the multimeric matrix form of VN but not the monomeric plasma form. Using various competitors, the interaction domains in Mac-1 and VN were localized. Mac-1-expressing but not untransfected Chinese hamster ovary cells adhered strongly on VN. Introduction of a GFFKR deletion in the alphaM subunit of Mac-1, which increases the constitutive activation of the integrin, led to increased adhesion on VN. Peripheral human blood neutrophils adhered and migrated on multimeric VN in a Mac-1-dependent manner. CONCLUSIONS: These results show that there is a specific integrin-affinity-regulated interaction between Mac-1 and the matrix form but not the plasma form of VN that may significantly participate in leukocyte adhesion and extravasation.

Intermolecular contact regions in urokinase plasminogen activator receptor.

The glycolipid-anchored urokinase-type plasminogen activator receptor (uPAR) is engaged in various signal transduction events related to cell adhesion, migration and proliferation. In this study, using phage display and peptide array techniques, we have identified several intermolecular contact regions of uPAR. Phage-displayed uPAR fragments bound to immobilized soluble uPAR on magnetic beads, revealing that regions uPAR-(7-28) and uPAR-(60-91) in domain I, uPAR-(101-121) in domain II and uPAR-(240-260) in domain III are possible uPAR-uPAR contact sites. Using peptide array, two additional sites could be identified, uPAR-(51-59) in domain I and uPAR-(144-155) in domain II. The putative uPAR-uPAR interaction sites are different from the previously identified uPA-binding sites. Functionally, peptides uPAR-(84-95) and uPAR-(240-248) could partially inhibit differentiated human U937 monocyte adhesion to vitronectin in the presence of uPA, indicating that these two uPAR regions might be involved not only in uPAR-uPAR but also in uPAR-vitronectin interactions. We propose that multiple uPAR-uPAR ectodomain interactions contribute considerably to the regulation of various cellular functions of uPAR.

Age-related endothelial dysfunction : potential implications for pharmacotherapy.

Aging per se is associated with abnormalities of the vascular wall linked to both structural and functional changes that can take place at the level of the extracellular matrix, the vascular smooth muscle and the endothelium of blood vessels. Endothelial dysfunction is generally defined as a decrease in the capacity of the endothelium to dilate blood vessels in response to physical and chemical stimuli. It is one of the characteristic changes that occur with age, independently of other known cardiovascular risk factors. This may account in part for the increased incidence of cardiovascular events in elderly people that can be reversed by restoring endothelial function. A better understanding of the mechanisms involved and the aetiopathogenesis of this process will help in the search for new therapeutic agents.Age-dependent alteration of endothelium-dependent relaxation seems to be a widespread phenomenon both in conductance and resistance arteries from several species. In the course of aging, there is an alteration in the equilibrium between relaxing and contracting factors released by the endothelium. Hence, there is a progressive reduction in the participation of nitric oxide and endothelium-derived hyperpolarising factor associated with increased participation of oxygen-derived free radicals and cyclo-oxygenase-derived prostanoids. Also, the endothelin-1 and angiotensin II pathways may play a role in age-related endothelial dysfunction. The use of drugs acting at different levels of these signalling cascades, including antioxidant therapy, lipid-lowering drugs and estrogens, seems to be promising.

Preservation of vascular contraction during ageing: dual effect on calcium handling and sensitization.

(1) The present study was aimed to characterize the effects of ageing on vascular contraction by noradrenaline in rat isolated arteries. The existence of vascular bed heterogeneity was investigated in endothelium-denuded conductance (aorta) and resistance (small mesenteric artery, SMA) arteries, with respect to Ca(2+) handling, Ca(2+) sensitization or Ca(2+)-independent mechanisms. (2) In both arteries, contractions to noradrenaline were not different between adult and aged rats. (3) In Ca(2+)- free medium, noradrenaline elicited a transient increase in tension that was reduced by the Ca(2+) mobilizing agents, ryanodine and thapsigargin, in arteries from adult rats. A loss of the thapsigargin- but not the ryanodine-sensitive component of noradrenaline-induced contraction was observed in the two arteries from aged rats. (4) After depletion of Ca(2+) stores with noradrenaline, addition of exogenous CaCl(2) produced a sustained contraction that was decreased to the same extent by the protein kinase C inhibitor, GF 109203X and the tyrosine kinase inhibitor, tyrphostin A-23, in arteries from adult and aged rats. The Rho-associated protein kinase inhibitor, Y-27632, caused identical relaxation of noradrenaline pre-contracted arteries from both age groups. (5) Basal intracellular calcium ([Ca(2+)](i)) was higher in SMA from aged than from adult rats. In addition, the noradrenaline [Ca(2+)](i)-force relationship was significantly shifted to the right in the SMA from aged rats. (6) Altogether, these data indicate that responsiveness to noradrenaline is preserved both in conductance and resistance arteries with ageing. The latter results from the association of increased basal [Ca(2+)](i), changes in Ca(2+) handling at the level of thapsigargin-sensitive sarcoplasmic reticulum Ca(2+)-ATPases and decreased myofilament sensitivity to Ca(2+).