Regulation of cholesterol homeostasis in macrophages and consequences for atherosclerotic lesion development

Foam cell formation due to excessive accumulation of cholesterol by macrophages is a pathological hallmark of atherosclerosis. Macrophages cannot limit the uptake of cholesterol and therefore depend on cholesterol efflux pathways for preventing their transformation into foam cells. Several ABC-transporters, including ABCA1 and ABCG1, facilitate the efflux of cholesterol from macrophages. These transporters, however, also affect membrane lipid asymmetry which may have important implications for cellular endocytotic pathways. We propose that in addition to the generally accepted role of these ABC-transporters in the prevention of foam cell formation by induction of cholesterol efflux from macrophages, they also influence the macrophage endocytotic uptake.     

Salicylate improves macrophage cholesterol homeostasis via activation of Ampk

Atherosclerosis stems from imbalances in lipid metabolism and leads to maladaptive inflammatory responses. The AMP-activated protein kinase (Ampk) is a highly conserved serine/threonine kinase that regulates many aspects of lipid and energy metabolism, although its specific role in controlling macrophage cholesterol homeostasis remains unclear. We sought to address this question by testing the effects of direct Ampk activators in primary bone marrow-derived macrophages from Ampk β1-deficient (β1^−/−) mice. Macrophages from Ampk β1^−/− mice had enhanced lipogenic capacity and diminished cholesterol efflux, although cholesterol uptake was unaffected. Direct activation of Ampk β1 via salicylate (the unacetylated form of aspirin) or A-769662 (a small molecule activator), decreased the synthesis of FAs and sterols in WT but not Ampk β1^−/− macrophages. In lipid-laden macrophages, Ampk activation decreased cholesterol content (foam cell formation) and increased cholesterol efflux to HDL and apoA-I, effects that occurred in an Ampk β1-dependent manner. Increased cholesterol efflux was also associated with increased gene expression of the ATP binding cassette transporters, Abcg1 and Abca1. Moreover, in vivo reverse cholesterol transport was suppressed in mice that received Ampk β1^−/− macrophages compared with the WT control. Our data highlight the therapeutic potential of targeting macrophage Ampk with new or existing drugs for the possible reduction in foam cell formation during the early stages of atherosclerosis.     

Inhibition of the NLRP3 inflammasome attenuates foam cell formation of THP-1 macrophages by suppressing ox-LDL uptake and promoting cholesterol efflux

The NOD-like receptor family, pyrin domain–containing protein 3 (NLRP3) inflammasome plays an important role in the development of atherosclerosis. The activated NLRP3 inflammasome has been reported to promote macrophage foam cell formation, but not all studies have obtained the same result, and how NLRP3 inflammasome is involved in the formation of foam cells remains elusive. We used selective NLRP3 inflammasome inhibitors and NLRP3-deficient THP-1 cells to assess the effect of NLRP3 inflammasome inhibition on macrophage foam cell formation, oxidized low-density lipoprotein (ox-LDL) uptake, esterification, and cholesterol efflux, as well as the expression of associated proteins. Inhibition of the NLRP3 inflammasome attenuated foam cell formation, diminished ox-LDL uptake, and promoted cholesterol efflux from THP-1 macrophages. Moreover, it downregulated CD36, acyl coenzyme A: cholesterol acyltransferase-1 and neutral cholesterol ester hydrolase expression; upregulated ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type I (SR-BI) expression; but had no effect on the expression of scavenger receptor class A and ATP-binding cassette transporter G1. Collectively, our findings show that inhibition of the NLRP3 inflammasome decreases foam cell formation of THP-1 macrophages via suppression of ox-LDL uptake and enhancement of cholesterol efflux, which may be due to downregulation of CD36 expression and upregulation of ABCA1 and SR-BI expression, respectively.     

LPS-induced suppression of macrophage cholesterol efflux is mediated by adipocyte enhancer-binding protein 1

Macrophages facilitate clearance of cholesterol from the body via reverse cholesterol transport (RCT). The first event in RCT is internalization of modified low density lipoprotein by macrophages, upon which PPARγ1 and LXRα signaling pathways are turned on, leading to the transactivation of a cascade of genes (e.g. ABCA1 and ABCG1), whose products promote macrophage cholesterol efflux. Down-regulation of macrophage cholesterol efflux mediators leads to an imbalance in cholesterol homeostasis, promoting foam cell formation. Lipopolysaccharide (LPS) has been shown to suppress PPARγ1 and its downstream target genes in macrophages, inducing foam cell formation; a key mechanism proposed to underlie bacterial infection-induced atherosclerosis. Herein, we show that adipocyte enhancer-binding protein 1 (AEBP1) is up-regulated during monocyte differentiation. Moreover, we provide experimental evidence suggesting that AEBP1 expression is induced by LPS, and that LPS-induced down-regulation of pivotal macrophage cholesterol efflux mediators, leading to foam cell formation, is largely mediated by AEBP1. Although AEBP1-independent pathways seem to contribute to these LPS effects, such pathways can only mediate lesser and delayed effects of LPS on macrophage cholesterol efflux and development of foam cells. We speculate that AEBP1 may serve as a potential therapeutic target for the prevention/treatment of bacterial infection-induced atherosclerosis.     

Excess Nitric Oxide Activates TRPV1-Ca2+-Calpain Signaling and Promotes PEST-dependent Degradation of Liver X Receptor α

Excess nitric oxide (NO) deregulates cholesterol metabolism in macrophage foam cells, yet the underlying molecular mechanism is incompletely understood. To investigate the mechanism, we found that in macrophages, treatment with NO donors S-nitroso-N-acetyl-D,L-penicillamine (SNAP) or diethylenetriamine/nitric oxide induced LXRα degradation and reduced the expression of the downstream target of LXRα, ATP-binding cassette transporter A1 (ABCA1), and cholesterol efflux. In addition, SNAP induced calcium (Ca²⁺) influx into cells, increased calpain activity and promoted the formation of calpain-LXRα complex. Pharmacological inhibition of calpain activity reversed the SNAP-induced degradation of LXRα, down-regulation of ABCA1 and impairment of cholesterol efflux in macrophages. SNAP increased the formation of calpain-LXRα complex in a Pro-Glu-Ser-Thr (PEST) motif-dependent manner. Truncation of the PEST motif in LXRα abolished the calpain-dependent proteolysis. Removal of extracellular Ca²⁺ by EGTA or pharmacological inhibition of TRPV1 channel activity diminished SNAP-induced increase in intracellular Ca²⁺, calpain activation, LXRα degradation, ABCA1 down-regulation and impaired cholesterol efflux. In conclusion, excess NO may activate calpain via TRPV1-Ca²⁺ signaling and promote the recognition of calpain in the PEST motif of LXRα, thereby leading to degradation of LXRα and, ultimately, downregulated ABCA1 expression and impaired ABCA1-dependent cholesterol efflux in macrophages.     

Neuropeptide Y and its receptor subtypes specifically modulate rat peritoneal macrophage functions in vitro: counter regulation through Y1 and Y2/5 receptors

It is well documented that neuropeptide Y (NPY) exerts a wide range of biological functions through at least five NPY Y receptor subtypes (Y1-Y5), but its immunological effects only recently came into focus. Using NPY family peptides and NPY-related receptor-specific peptides as well as Y1 and Y2 receptor antagonists, we have tested which NPY Y receptors are involved in NPY-induced modulation of rat peritoneal macrophage function in vitro. NPY and PYY increased oxidative burst in phorbol myristate acetate (PMA)-stimulated macrophages involving activation of protein kinase C (PKC), and decreased it in zymosan-stimulated cells resembling inhibition of signaling pathways subsequent to binding of zymosan particles for the iC3b fragment receptor on macrophages. The combined treatment with NPY and NPY Y receptor antagonists revealed that NPY-induced potentiation of oxidative burst in PMA-stimulated cells is mediated through Y1 and Y2 receptors, while NPY-induced suppression in zymosan-stimulated cells is mediated through Y2 receptors only. NPY-related peptides differently modulated macrophage function, confirming involvement of NPY Y2 receptor in both potentiation and suppression of oxidative burst in these cells. Additionally, it was shown that NPY Y5 receptor mediated suppression of oxidative burst in PMA- and zymosan-stimulated macrophages. Taken together, the present data reveal an NPY Y1 and Y2/Y5 receptor interaction in NPY-induced modulation of macrophage functions related to inflammation.     

Over-activation of NMDA receptors promotes ABCA1 degradation and foam cell formation

ATP-binding cassette transporter A1 (ABCA1) is an essential regulator of intracellular cholesterol efflux. Secreted cholesterol binds to lipid-free apolipoprotein A-I (apoA-I) in peripheral blood to constitute high-density lipoprotein cholesterol (HDL) complexes. ABCA1 protein on the surface of macrophages acts as a crucial controller in preventing cholesterol accumulation. Importantly, ABCA1 is unstable and easily degraded via a series of biochemical activities, including but not limited to calpain-mediated and ubiquitin-proteasome system-mediated processes. How accelerated ABCA1 degradation impacts disordered lipid metabolism in macrophages and foam cell formation is unclear. N-methyl d-aspartate receptors (NMDARs) are ionotropic glutamate receptors with high calcium permeability. Calcium influx via NMDARs activates downstream signaling pathways. Over-activation of NMDARs stimulated by NMDA contributes to dysfunctional lipid metabolism in macrophages and foam cell formation via promotion of calpain-mediated ABCA1 proteolysis. However, increased NMDAR activity does not affect liver X receptor expression or ABCA1 mRNA levels. Following NMDA receptor silencing or calpain inhibition, NMDA treatment did not reduce ABCA1 protein levels, nor caused lipid accumulation in macrophages. In addition, NMDAR over-activation activates NF-κB signaling to promote IL-1β and IL-6 macrophage marker expression. However, NMDAR silencing and calpain inhibition reduce inflammatory macrophage responses. In summary, our study suggests that NMDAR activation reduces surface ABCA1 protein, promotes lipid accumulation, and induces the production and secretion of many inflammatory mediators in macrophages, possibly through enhanced calpain-mediated ABCA1 protein degradation. Thus, the NMDAR receptor may be a novel pharmacologic target for atherosclerosis therapy.     

Interleukin-10 Facilitates Both Cholesterol Uptake and Efflux in Macrophages

Foam cell formation is a hallmark event during atherosclerosis. The current paradigm is that lipid uptake by scavenger receptor in macrophages initiates the chronic proinflammatory cascade and necrosis core formation that characterize atherosclerosis. We report here that a cytokine considered to be anti-atherogenic, interleukin-10 (IL10), promotes cholesterol uptake from modified lipoproteins in macrophages and its transformation into foam cells by increasing the expression of scavenger receptor CD36 and scavenger receptor A. Although uptake of modified lipoproteins is considered proatherogenic, we found that IL10 also increases cholesterol efflux from macrophages to protect against toxicity of free cholesterol accumulation in the cell. This process was PPARγ-dependent and was mediated through up-regulation of ABCA1 (ATP-binding cassette transporter A1) protein expression. Importantly, expression of inflammatory molecules, such as tumor necrosis factor-α, intercellular adhesion molecule-1, and MMP9 as well as apoptosis were dramatically suppressed in lipid-laden foam cells treated with IL10. The notion that IL10 can mediate both the uptake of cholesterol from modified lipoproteins and the efflux of stored cholesterol suggests that the process of foam cell formation is not necessarily detrimental as long as mechanisms of cholesterol efflux and transfer to an exogenous acceptor are functioning robustly. Our results present a comprehensive antiatherogenic role of IL10 in macrophages, including enhanced disposal of harmful lipoproteins, inhibition of inflammatory molecules, and reduced apoptosis.     

Neuropeptide Y modulates functions of inflammatory cells in the rat: distinct role for Y1, Y2 and Y5 receptors

Neuropeptide Y (NPY) has been reported to be a potent anti-inflammatory peptide with ability to directly modulate activity of granulocytes and macrophages. The present study aimed to correlate the effects of NPY in vivo on lipopolysaccharide-induced air-pouch exudates cells and in vitro on peripheral blood leukocytes functions. The role of different Y receptors was examined using NPY-related peptides and antagonists with diverse subtype specificity and selectivity for Y receptors. Y1, Y2 and Y5 receptors were detected on air-pouch exudates cells (flow cytometry) and peripheral blood granulocytes (immunocitochemistry). NPY in vivo reduced inflammatory cells accumulation into the air pouch, and decreased their adherence and phagocytic capacity via Y2/Y5 and Y1/Y2 receptors, respectively. Quite the opposite, NPY in vitro potentiated adhesiveness and phagocytosis of peripheral blood granulocytes and monocytes by activating Y1 receptor. The differences between in vivo and in vitro effects of NPY on rat inflammatory cells functions are mostly due to dipeptidyl peptidase 4 activity. In addition, suppressive effect of NPY in vivo is highly dependent on the local microenvironment, peptide truncation and specific Y receptors interplay.     

Macrophage‐derived exosomal miR‐4532 promotes endothelial cells injury by targeting SP1 and NF‐κB P65 signalling activation

Atherosclerosis is a complex pathological process involving macrophages, endothelial cells and vascular smooth muscle cells that can lead to ischemic heart disease; however, the mechanisms underlying cell‐to‐cell communication in atherosclerosis are poorly understood. In this study, we focused on the role of exosomal miRNAs in crosstalk between macrophages and endothelial cells and explored the rarely studied molecular mechanisms involved. Our in vitro result showed that macrophage‐derived exosomal miR‐4532 significantly disrupted human umbilical vein endothelial cells (HUVECs) function by targeting SP1 and downstream NF‐κB P65 activation. In turn, increased endothelin‐1 (ET‐1), intercellular cell adhesion molecule‐1 (ICAM‐1) and vascular cell adhesion molecule‐1 (VCAM‐1) and decreased endothelial nitric oxide synthase (eNOS) expression in HUVECs increased attraction of macrophages, exacerbating foam cell formation and transfer of exosomal miR‐4532 to HUVECs. MiR‐4532 overexpression significantly promoted endothelial injury and pretreatment with an inhibitor of miR‐4532 or GW4869 (exosome inhibitor) could reverse this injury. In conclusion, our data reveal that exosomes have a critical role in crosstalk between HUVECs and macrophages. Further, exosomal miR‐4532 transferred from macrophages to HUVECs and targeting specificity protein 1 (SP1) may be a novel therapeutic target in patients with atherosclerosis.     

Cloning of cDNAs with PDCD2C domain and their expressions during apoptosis of HEK293T cells

Modulation of the expression of genes involved in the control of cholesterol homeostasis by sterols in macrophages is crucial to foam cell formation. To characterize this regulation in THP-1 macrophages, we examined the effect of sterol loading and unloading on the expression of a number of genes that participate in lipoprotein uptake and cholesterol efflux. Sterol loading by exposure to acetylated LDL for 24 h resulted in an increase in free and esterified cholesterol of 1.4 and 1.8-fold, respectively. Under these conditions, the mRNA levels for SR-A were reduced a 59%, while those of CYP27 were increased by 4.6-fold. However, the expression of other genes involved in cholesterol efflux (ABCA1, ABCG1 and CLA-1) was not modified, despite a high intracellular cholesterol accumulation specially in the form of esterified cholesterol.On the other hand, HDL exposure reduced intracellular cholesterol content to 70%, and caused an increase in the expression of CD36 (78%), SR-A (51%) and CLA-1 (136%). Conversely, the expression of ABCA1, ABCG1 and CYP27 was decreased by 49, 67 and 57%, respectively. These findings indicate that in THP-1 macrophages, the expression of genes for receptors involved in lipoprotein binding and uptake tends to decrease upon cholesterol loading and to increase by cholesterol depletion, while the opposite pattern is found regarding the mRNA levels for proteins involved in cholesterol efflux.     

Reduction in ABCG1 in Type 2 diabetic mice increases macrophage foam cell formation

Atherosclerosis development is accelerated severalfold in patients with Type 2 diabetes. In the initial stages of disease, monocytes transmigrate into the subendothelial space and differentiate into foam cells. Scavenger receptors and ATP binding cassette (ABC) Transporters play an important role in foam cell formation as they regulate the influx and efflux of oxidized lipids. Here, we show that peritoneal macrophages isolated from Type 2 diabetic db/db mice have decreased expression of the ABC transporter ABCG1 and increased expression of the scavenger receptor CD36. We found a 2-fold increase in accumulation of esterified cholesterol in diabetic db/db macrophages compared with wild-type control macrophages. Diabetic db/db macrophages also had impaired cholesterol efflux to high density lipoprotein but not to lipid-free apo A-I, suggesting that the increased esterified cholesterol in diabetic db/db macrophages was due to a selective loss of ABCG1-mediated efflux to high density lipoprotein. Additionally, we were able to confirm down-regulation of ABCG1 using C57BL/6J peritoneal macrophages cultured in elevated glucose in vitro (25 mM glucose for 7 days), suggesting that ABCG1 expression in diabetic macrophages is regulated by chronic exposure to elevated glucose. Diabetic KK(ay) mice were also studied and were found to have decreased ABCG1 expression without an increase in CD36. These observations demonstrate that ABCG1 plays a major role in macrophage cholesterol efflux and that decreased ABCG1 function can facilitate foam cell formation in Type 2 diabetic mice.     

ABCG1 mediated oxidized LDL-derived oxysterol efflux from macrophages

Objectives

The uptake of oxidized LDL (oxLDL) by macrophages is a key initial event in atherogenesis, and the removal of oxidized lipids from artery wall via reverse cholesterol transport is considered antiatherogenic. The aims of this study were to investigate the pathways mediating the removal of oxysterols from oxLDL-loaded macrophages, and the subsequent uptake of the oxysterols by hepatocytes.

Methods

LDL was labeled with [3H]cholesterol, and LDL-[3H]cholesterol was oxidized by copper using a standard method. [3H]oxysterol formation in oxLDL was analyzed by thin layer chromatography. oxLDL-[3H]sterol was incubated with macrophages, allowing the uptake of [3H]sterol by macrophages. [3H]sterol efflux from macrophages mediated by ATP binding cassette transporters (ABCA1, ABCG1), or scavenger receptor class B type I (SR-BI) was measured. The subsequent uptake of the [3H]sterol by hepatocytes was also determined.

Results

7-Ketocholesterol was the major oxysterol formed in oxLDL, and it was significantly higher in oxLDL compared with that in native LDL (naLDL). oxLDL-derived sterol efflux to HDL from macrophages was significantly increased compared with naLDL-derived sterol, and it was mainly mediated by ABCG1, but not by ABCA1 or SR-BI. Moreover, although HDL dose-dependently induced sterol efflux from macrophages, only the exported sterol by ABCG1 pathway was efficiently taken up by hepatocytes.

Conclusions

ABCG1 mediates oxysterol efflux from oxLDL-loaded macrophages, and the exported oxysterol by ABCG1 pathway can be selectively taken up by hepatocytes.     

Anti-atherogenic mechanisms of high density lipoprotein: Effects on myeloid cells

In some settings increasing high density lipoprotein (HDL) levels has been associated with a reduction in experimental atherosclerosis. This has been most clearly seen in apolipoprotein A-I (apoA-I) transgenic mice or in animals infused with HDL or its apolipoproteins. A major mechanism by which these treatments are thought to delay progression or cause regression of atherosclerosis is by promoting efflux of cholesterol from macrophage foam cells. In addition, HDL has been described as having anti-inflammatory and other beneficial effects. Some recent research has linked anti-inflammatory effects to cholesterol efflux pathways but likely multiple mechanisms are involved. Macrophage cholesterol efflux may have a role in facilitating emigration of macrophages from lesions during regression. While macrophages can mediate cholesterol efflux by several pathways, studies in knockout mice or cells point to the importance of active efflux mediated by ATP binding cassette transporter (ABC) A1 and G1. In addition to traditional roles in macrophages, these transporters have been implicated in the control of hematopoietic stem cell proliferation, monocytosis and neutrophilia, as well as activation of monocytes and neutrophils. Thus, HDL and cholesterol efflux pathways may have important anti-atherogenic effects at all stages of the myeloid cell/monocyte/dendritic cell/macrophage lifecycle. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Apolipoprotein B stimulates formation of monocyte-macrophage surface-connected compartments and mediates uptake of low density lipoprotein-derived liposomes into these compartments

Much of the cholesterol that accumulates in atherosclerotic plaques is found within monocyte-macrophages transforming these cells into "foam cells." Native low density lipoprotein (LDL) does not cause foam cell formation. Treatment of LDL with cholesterol esterase converts LDL into cholesterol-rich liposomes having >90% cholesterol in unesterified form. Similar cholesterol-rich liposomes are found in early developing atherosclerotic plaques surrounding foam cells. We now show that cholesterol-rich liposomes produced from cholesterol esterase-treated LDL can cause human monocyte-macrophage foam cell formation inducing a 3-5-fold increase in macrophage cholesterol content of which >60% is esterified. Although cytochalasin D inhibited LDL liposome-induced macrophage cholesteryl ester accumulation, LDL liposomes did not enter macrophages by phagocytosis. Rather, the LDL liposomes induced and entered surface-connected compartments within the macrophages, a unique endocytic pathway in these cells that we call patocytosis. LDL liposome apoB rather than LDL liposome lipid mediated LDL liposome uptake by macrophages. This was shown by the findings that: 1) protease treatment of the LDL liposomes prevented macrophage cholesterol accumulation; 2) liposomes prepared from LDL lipid extracts did not cause macrophage cholesterol accumulation; and 3) purified apoB induced and accumulated within macrophage surface-connected compartments. Although apoB mediated the macrophage uptake of LDL liposomes, this uptake did not occur through LDL, LDL receptor-related protein, or scavenger receptors. Also, LDL liposome uptake was not sensitive to treatment of macrophages with trypsin or heparinase. Cholesterol esterase-mediated transformation of LDL into cholesterol-rich liposomes is an LDL modification that: 1) stimulates uptake of LDL cholesterol by apoB-dependent endocytosis into surface-connected compartments, and 2) causes human monocyte-macrophage foam cell formation.     

Wogonin promotes cholesterol efflux by increasing protein phosphatase 2B-dependent dephosphorylation at ATP-binding cassette transporter-A1 in macrophages

Wogonin, one component in Scutellaria baicalensis Georgi extracts, has several beneficial properties for cancers and inflammatory diseases. However, the efficacy of wogonin in cholesterol metabolism of macrophages remains unknown. In macrophages, cholesterol uptake is controlled by scavenger receptors (SR-A and CD36) and cholesterol efflux by SR-BI, ATP-binding cassette transporter-A1 (ABCA1) and ABCG1. In the present study, we investigated the effect and underlying molecular mechanism of wogonin on the formation of macrophage foam cells by murine J774.A1 macrophages. Wogonin attenuated oxidized low-density lipoprotein (oxLDL)-induced cholesterol accumulation in macrophages. The binding of oxLDL to macrophages and protein expression of SR-A and CD36 were not affected by wogonin. Wogonin enhanced cholesterol efflux and increased the protein level of ABCA1 without affecting the protein expression of SR-BI or ABCG1. Inhibition of ABCA1 by pharmacological inhibitor 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid disodium salt or neutralizing antibody abolished this suppressive effect of wogonin on lipid accumulation. Moreover, the up-regulation of ABCA1 protein by wogonin resulted from a decrease in degradation rate of ABCA1 protein, with no effect on ABCA1 mRNA expression. This reduction in ABCA1 degradation was due to increased protein phosphatase 2B (PP2B)-mediated ABCA1 dephosphorylation, as evidenced by increased interaction between ABCA1 and PP2B; pharmacological inhibition of PP2B would prevent wogonin-induced ABCA1 protein expression, dephosphorylation and attenuation of lipid accumulation. Collectively, wogonin increases the protein stability of ABCA1 via PP2B-mediated dephosphorylation, thus leading to reduced cholesterol accumulation in macrophage foam cells.     

Role of Kir2.1 in human monocyte‐derived foam cell maturation

The role of K⁺ channels in macrophage immunomodulation has been well‐established. However, it remains unclear whether K⁺ channels are involved in the lipid uptake of macrophages. The expression and function of the inward rectifier potassium channel (Kir2.1, KCNJ2) in Human acute monocytic leukemia cell line (THP‐1) cells and human monocytes derived macrophages (HMDMs) were investigated using RT‐PCR and western blotting, and patch clamp technique. The expression of scavenger receptors in THP‐1–derived macrophages was detected using western blotting. Expressions of Kir2.1 mRNA and protein in HMDMs were significantly decreased by 60% (P < 0.05) and 90% (P < 0.001) on macrophage maturation, but overexpressed by approximately 1.3 (P > 0.05) and 3.8 times (P = 0.001) after foam cell formation respectively. Concurrently, the Kir2.1 peak current density in HMDMs, mature macrophages and foam cells, measured at −150 mV, were −22.61 ± 2.1 pA/pF, −7.88 ± 0.60 pA/pF and −13.39 ± 0.80 pA/pF respectively (P < 0.05). In association with an up‐regulation of Kir2.1 in foam cells, the SR‐A protein level was significantly increased by over 1.5 times compared with macrophages (P < 0.05). THP‐1 cells contained much less lipids upon Kir2.1 knockdown and cholesterol ester/total cholesterol ratio was 29.46 ± 2.01% (P < 0.05), and the SR‐BI protein level was increased by over 6.2 times, compared to that of macrophages (P < 0.001). Kir2.1 may participate in macrophage maturation and differentiation, and play a key role in lipid uptake and foam cell formation through modulating the expression of scavenger receptors.     

Novel role of Y1 receptors in the coordinated regulation of bone and energy homeostasis

The importance of neuropeptide Y (NPY) and Y2 receptors in the regulation of bone and energy homeostasis has recently been demonstrated. However, the contributions of the other Y receptors are less clear. Here we show that Y1 receptors are expressed on osteoblastic cells. Moreover, bone and adipose tissue mass are elevated in Y1(-/-) mice with a generalized increase in bone formation on cortical and cancellous surfaces. Importantly, the inhibitory effects of NPY on bone marrow stromal cells in vitro are absent in cells derived from Y1(-/-) mice, indicating a direct action of NPY on bone cells via this Y receptor. Interestingly, in contrast to Y2 receptor or germ line Y1 receptor deletion, conditional deletion of hypothalamic Y1 receptors in adult mice did not alter bone homeostasis, food intake, or adiposity. Furthermore, deletion of both Y1 and Y2 receptors did not produce additive effects in bone or adiposity. Thus Y1 receptor pathways act powerfully to inhibit bone production and adiposity by nonhypothalamic pathways, with potentially direct effects on bone tissue through a single pathway with Y2 receptors.     

Endothelin-1 exacerbates lipid accumulation by increasing the protein degradation of the ATP-binding cassette transporter G1 in macrophages

Endothelin-1 (ET-1), a potent proatherogenic vasoconstrictive peptide, is known to promote macrophage foam cell formation via mechanisms that are not fully understood. Excessive lipid accumulation in macrophages is a major hallmark during the early stages of atherosclerotic lesions. Cholesterol homeostasis is tightly regulated by scavenger receptors (SRs) and ATP-binding cassette (ABC) transporters during the transformation of macrophage foam cells. The aim of this study was to investigate the possible mechanisms by which ET-1 affects lipid accumulation in macrophages. Our results demonstrate that oxidized low-density lipoprotein (oxLDL) treatment increases lipid accumulation in rat bone marrow-derived macrophages. Combined treatment with ET-1 and oxLDL significantly exacerbated lipid accumulation in macrophages as compared to treatment with oxLDL alone. The results of Western blotting show that ET-1 markedly decreased the ABCG1 levels via ET type A and B receptors and activation of the phosphatidylinositol 3-kinase pathway; however, ET-1 had no effect on the protein expression of CD36, SR-BI, SR-A, or ABCA1. In addition, real-time PCR analysis showed that ET-1 treatment did not affect ABCG1 mRNA expression. We also found that ET-1 decreases ABCG1 possibly due to the enhancement of the proteosome/calpain pathway-dependent degradation of ABCG1. Moreover, ET-1 significantly reduced the efficiency of the cholesterol efflux in macrophages. Taken together, these findings suggest that ET-1 may impair cholesterol efflux and further exacerbate lipid accumulation during the transformation of macrophage foam cells.     

Stress, NPY and vascular remodeling: Implications for stress-related diseases

Neuropeptide Y (NPY) has long been known to be involved in stress, centrally as an anxiolytic neuromodulator, and peripherally as a sympathetic nerve- and in some species, platelet-derived vasoconstrictor. The peptide is also a vascular mitogen, via Y1/Y5, and is angiogenic via Y2/Y5 receptors. Arterial injury activates platelet NPY and vascular Y1 receptors, inducing medial hypertrophy and neointima formation. Exogenous NPY, dipeptidyl peptidase IV (DPPIV, forming an Y2/Y5-selective agonist) and chronic stress augment these effects and occlude vessels with atherosclerotic-like lesions, containing thrombus and lipid-laden macrophages. Y1 antagonist blocks stress-induced vasoconstriction and post-angioplasty occlusions, and hence may be therapeutic in angina and atherosclerosis/restenosis. Conversely, tissue ischemia activates neuronal and platelet-derived NPY, Y2/Y5 and DPPIV, which stimulate angiogenesis/arteriogenesis. NPY-Y2-DPPIV agonists may be beneficial for ischemic revascularization and wound healing, whereas antagonists may be therapeutic in retinopathy, tumors, and obesity. Since stress is an underestimated risk factor in many of these conditions, NPY-based drugs may offer new treatment possibilities.