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1.
Biomedicines ; 11(10)2023 Oct 05.
Article in English | MEDLINE | ID: mdl-37893077

ABSTRACT

GCN2 is one of the main sensors of amino acid starvation stress, and its activation in the stressful tumor microenvironment plays a crucial role in tumor survival and progression. We hypothesized that elevated polyamine biosynthesis and subsequent depletion of precursor arginine activates GCN2, thus rewiring metabolism to support tumor cell survival and drive myeloid immunosuppressive function. We sought to determine if the anti-tumor efficacy of a polyamine blocking therapy (PBT) may be mediated by its effect on GCN2. Unlike wild-type mice, PBT treatment in GCN2 knockout mice bearing syngeneic B16.F10 or EG7 tumors resulted in no tumor growth inhibition and no changes in the profile of infiltrating tumor immune cells. Studies with murine bone marrow cell cultures showed that increased polyamine metabolism and subsequent arginine depletion and GCN2 activation played an essential role in the generation and cytoprotective autophagy of myeloid derived suppressor cells (MDSCs) as well as the M2 polarization and survival of macrophages, all of which were inhibited by PBT. In all, our data suggest that polyamine-dependent GCN2 signaling in stromal cells promotes tumor growth and the development of the immunosuppressive tumor microenvironment, and that the PBT anti-tumor effect is mediated, at least in part, by targeting GCN2.

2.
Mol Carcinog ; 61(6): 527-536, 2022 06.
Article in English | MEDLINE | ID: mdl-35338515

ABSTRACT

Coagulation proteases and the generation of thrombin are increased in tumors. In addition, chemotherapeutic agents commonly used to treat malignant cancers can exacerbate cancer-associated thromboses. Thrombin can modify tumor cell behavior directly through the activation of protease-activated receptors (PAR) or indirectly by generating fibrin matrices. In addition to its role in generating fibrin to promote hemostasis, thrombin acts directly on multiple effector cells of the immune system impacting both acute and chronic inflammatory processes. Thrombin-mediated release of interleukin-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1 leads to the accumulation of multiple tumor-infiltrating immunosuppressive cell populations including myeloid derived suppresser cells, M2-like macrophages, and T regulatory cells. Ablation of PAR-1 from the tumor microenvironment, but not the tumor, has been shown to dramatically reduce tumor growth and metastasis in multiple tumor models. Thrombin-activated platelets release immunosuppressive cytokines including transforming growth factor-ß that can inhibit natural killer cell activity, helping tumor cells to evade host immunosurveillance. Taken together, there is strong evidence that thrombin influences cancer progression via multiple mechanisms, including the tumor immune response, with thrombin emerging as a target for novel therapeutic strategies for cancer.


Subject(s)
Neoplasms , Thrombin , Fibrin , Humans , Immunity , Neoplasms/pathology , Receptor, PAR-1/physiology , Thrombin/pharmacology , Tumor Microenvironment
3.
Cancer Biol Ther ; 22(3): 225-237, 2021 03 04.
Article in English | MEDLINE | ID: mdl-33602034

ABSTRACT

BRAF mutations are present in over half of all melanoma tumors. Although BRAF inhibitors significantly improve survival of patients with metastatic melanoma, recurrences occur within several months. We previously reported that BRAF mutant melanoma cells are more sensitive to a novel arylmethyl-polyamine (AP) compound that exploits their increased polyamine uptake compared to that of BRAF wildtype cells. Using an animal model of BRAF inhibitor-resistant melanoma, we show that co-treatment with the BRAF inhibitor, PLX4720, and AP significantly delays the recurrence of PLX4720-resistant melanoma tumors and decreases tumor-promoting macrophages. Development of BRAF inhibitor-resistance enriches for metastatic cancer stem cells (CSC) and increases tumor-promoting macrophages. In vitro studies demonstrated that CD304+, CXCR4+ spheroid cultures of BRAF mutant melanoma cells are resistant to PLX4720 but are more sensitive to AP compared to monolayer cultures of the same cells. AP significantly inhibited YUMM1.7 melanoma cell invasiveness across a Matrigel-coated filter using the CXCR4 ligand, SDF-1α, as the chemoattractant. AP also blocked the chemotactic effect of SDF-1α on CXCR4+ macrophages and inhibited M2 polarization of macrophages. In melanoma-macrophage co-cultures, AP prevented the PLX4720-induced release of pro-tumorigenic growth factors, such as VEGF, from macrophages and prevented the macrophage rescue of BRAF mutant melanoma cells treated with PLX4720. Our study offers a novel therapy (AP) to treat chemo-resistant melanoma. AP is unique because it targets the polyamine transport system in BRAF inhibitor-resistant CSCs and also blocks CXCR4 signaling in invasive melanoma cells and pro-tumorigenic macrophages.


Subject(s)
Drug Resistance, Neoplasm/genetics , Melanoma/genetics , Polyamines/therapeutic use , Proto-Oncogene Proteins B-raf/metabolism , Animals , Disease Models, Animal , Humans , Mice , Mutation , Polyamines/pharmacology
4.
Mol Cancer Ther ; 19(10): 2012-2022, 2020 10.
Article in English | MEDLINE | ID: mdl-32747421

ABSTRACT

Despite unprecedented advances in the treatment of cancer through the use of immune checkpoint blockade (ICB), responses are not universal and alternative strategies are needed to enhance responses to ICB. We have shown previously that a novel polyamine blocking therapy (PBT), consisting of cotreatment with α-difluoromethylornithine (DFMO) to block polyamine biosynthesis and a Trimer polyamine transport inhibitor, decreases myeloid-derived suppressor cells (MDSC) and M2-like tumor-associated macrophages (TAM). Both MDSCs and TAMs promote tumor progression, inhibit antitumor immunity, and limit the efficacy of ICB. In this study, we investigated the use of PBT to heighten therapeutic responses to PD-1 blockade in mice bearing 4T1 mammary carcinoma and B16F10 melanoma tumors. Whereas PBT inhibited primary tumor growth in both tumor models, 4T1 lung metastases were also dramatically decreased in mice treated with PBT. Reductions in MDSC and TAM subpopulations in 4T1 tumors from PBT-treated mice were accompanied by reduced cytoprotective autophagy only in tumor-infiltrating MDSC and macrophage subpopulations but not in the lung or spleen. PBT treatment blunted M2-like alternative activation of bone marrow-derived macrophages and reduced STAT3 activation in MDSC cultures while increasing the differentiation of CD80+, CD11c+ macrophages. PBT significantly enhanced the antitumor efficacy of PD-1 blockade in both 4T1 and B16F10 tumors resistant to anti-PD-1 monotherapy, increasing tumor-specific cytotoxic T cells and survival of tumor-bearing animals beyond that with PBT or PD-1 blockade alone. Our results suggest that cotreatment with DFMO and the Trimer polyamine transport inhibitor may improve the therapeutic efficacy of immunotherapies in patients with cancer with resistant tumors.


Subject(s)
Antineoplastic Agents/therapeutic use , Myeloid Cells/metabolism , Polyamines/antagonists & inhibitors , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Animals , Antineoplastic Agents/pharmacology , Female , Humans , Mice
5.
Carcinogenesis ; 40(8): 1042-1051, 2019 08 22.
Article in English | MEDLINE | ID: mdl-31190067

ABSTRACT

Tumor promotion is strongly associated with inflammation and increased polyamine levels. Our understanding of relevant mechanisms responsible for arsenic-induced cancer remains limited. Previous studies suggest that arsenic targets and dysregulates stem cell populations that remain dormant in the skin until promoted to be recruited out of the bulge stem cell region, thus giving rise to skin tumors. In this study, we explored a possible mechanism by which increased keratinocyte polyamine biosynthesis promotes tumorsphere formation and invasiveness of arsenic-transformed HaCaT keratinocytes (As-HaCaT). Unlike parental HaCaT cells, As-HaCaT cells were tumorigenic in athymic nude mice, and the CD45negative epithelial tumor cells had enriched expression of Toll-Like Receptor 4 (TLR4), CD34 and CXCR4 as did As-HaCaT tumorsphere cultures compared to As-HaCaT monolayer cultures. Ornithine decarboxylase (ODC) overexpressing keratinocytes (Ker/ODC) release increased levels of the alarmin high mobility group box 1 (HMGB1). Ker/ODC conditioned medium (CM) stimulated As-HaCaT but not parental HaCaT tumorsphere formation, and this was inhibited by glycyrrhizin, an inhibitor of HMGB1, and by TAK242, an inhibitor of the HMGB1 receptor TLR4. Compared to parental HaCaT cells, As-HaCaT cells demonstrated greater invasiveness across a Matrigel-coated filter using either fibroblast CM or SDF-1α as chemoattractants. Addition of Ker/ODC CM or HMGB1 dramatically increased As-HaCaT invasiveness. Glycyrrhizin and TAK242 inhibited this Ker/ODC CM-stimulated invasion of As-HaCaT cells but not HaCaT cells. These results show that polyamine-dependent release of HMGB1 promotes the expansion of stem cell-like subpopulations in arsenic-transformed keratinocytes while also increasing their invasiveness, suggesting that polyamines may be a potential therapeutic target for the prevention and treatment of arsenic-initiated skin cancers.


Subject(s)
Arsenic/toxicity , Carcinogenesis/drug effects , HMGB1 Protein/genetics , Skin Neoplasms/genetics , Animals , Carcinogenesis/genetics , Cell Proliferation/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Glycyrrhizic Acid/pharmacology , Humans , Keratinocytes/drug effects , Keratinocytes/pathology , Mice , Mice, Nude , Neoplasm Invasiveness/genetics , Neoplasm Invasiveness/pathology , Polyamines/metabolism , Signal Transduction/drug effects , Skin/pathology , Skin Neoplasms/chemically induced , Skin Neoplasms/pathology , Toll-Like Receptor 4/genetics
6.
Oncotarget ; 8(48): 84140-84152, 2017 Oct 13.
Article in English | MEDLINE | ID: mdl-29137411

ABSTRACT

Most tumors maintain elevated levels of polyamines to support their growth and survival. This study explores the anti-tumor effect of polyamine starvation via both inhibiting polyamine biosynthesis and blocking the upregulated import of polyamines into the tumor. We demonstrate that polyamine blockade therapy (PBT) co-treatment with both DFMO and a novel polyamine transport inhibitor, Trimer PTI, significantly inhibits tumor growth more than treatment with DFMO or the Trimer PTI alone. The anti-tumor effect of PBT was lost in mice where CD4+ and CD8+ T cells were antibody depleted, implying that PBT stimulates an anti-tumor immune effect that is T-cell dependent. The PBT anti-tumor effect was accompanied by an increase in granzyme B+, IFN-γ+ CD8+ T-cells and a decrease in immunosuppressive tumor infiltrating cells including Gr-1+CD11b+ myeloid derived suppressor cells (MDSCs), CD4+CD25+ Tregs, and CD206+F4/80+ M2 macrophages. Stimulation with tumor-specific peptides elicited elevated antigen-specific IFN-γ secretion in splenocytes from PBT-treated mice, indicating that PBT treatment stimulates the activation of T-cells in a tumor-specific manner. These data show that combined treatment with both DFMO and the Trimer PTI not only deprives polyamine-addicted tumor cells of polyamines, but also relieves polyamine-mediated immunosuppression in the tumor microenvironment, thus allowing the activation of tumoricidal T-cells.

7.
Oncotarget ; 7(51): 85291-85305, 2016 Dec 20.
Article in English | MEDLINE | ID: mdl-27852034

ABSTRACT

Cancer is often associated with an increased risk of thrombotic complications which can be aggravated by treatment with chemotherapeutics such as cisplatin. Multiple lines of evidence suggest that thrombin activity promotes tumor growth and metastasis. We examined the effect of co-treatment with dabigatran etexilate, a direct thrombin inhibitor, and cisplatin using the murine ID8 ovarian cancer model. Mice receiving co-treatment with both dabigatran etexilate and low dose cisplatin had significantly smaller tumors, developed less ascites and had lower levels of circulating activated platelets and tissue factor (TF) positive microparticles than those treated with dabigatran etexilate or cisplatin alone. Co-treatment with dabigatran etexilate and cisplatin significantly decreased the number of Gr1+/CD11b+ myeloid derived suppresser cells and CD11b+/CD11c+ dendritic cells in the ascites of ID8 tumor-bearing mice. Co-treatment also significantly reduced levels of pro-tumorigenic cytokines including TGF-ß, VEGF, IL-6, IL-10, and MCP-1 in the ascites while increasing IFN-γ production by CD8+ effector T cells in the tumor ascites. These results demonstrate that co-treatment with dabigatran etexilate significantly augments the anti-tumor activity of cisplatin in ovarian tumor progression by alleviating the immunosuppressive microenvironment, suggesting that thrombin may be a potential therapeutic target for treatment of ovarian cancer.


Subject(s)
Antithrombins/therapeutic use , Blood Platelets/immunology , Cisplatin/therapeutic use , Dabigatran/therapeutic use , Dendritic Cells/immunology , Myeloid-Derived Suppressor Cells/immunology , Ovarian Neoplasms/drug therapy , Animals , CD8-Positive T-Lymphocytes/immunology , Carcinogenesis , Cell Growth Processes , Cells, Cultured , Cytokines/metabolism , Cytotoxicity, Immunologic , Drug Synergism , Drug Therapy, Combination , Female , Humans , Immunosuppression Therapy , Mice , Mice, Inbred C57BL , Ovarian Neoplasms/immunology , Tumor Burden , Tumor Microenvironment/drug effects , Xenograft Model Antitumor Assays
8.
Cancer Biol Ther ; 16(12): 1802-11, 2015.
Article in English | MEDLINE | ID: mdl-26383051

ABSTRACT

Cancer is often associated with an increased risk of thrombotic events which are exacerbated by treatment with chemotherapeutics such as cyclosphosphamide (CP). Evidence suggests that thrombin can stimulate tumor progression via formation of fibrin and activation of protease-activated receptors (PARs) and platelets. We examined the effect of co-treatment with CP and dabigatran etexilate, a direct inhibitor of thrombin, using the murine orthotopic 4T1 tumor model. Mice receiving co-treatment with both low dose CP and dabigatran etexilate had significantly smaller mammary tumors and fewer lung metastases than mice treated with CP or dabigratran etexilate alone. Co-treatment with dabigatran etexilate and low dose CP also significantly decreased the number of arginase(+)Gr-1(+)CD11b(+) myeloid derived suppressor cells as well as levels of TGF-ß in spleens from tumor bearing mice. 4T1 tumors express procoagulant tissue factor (TF) and spontaneously release TF(+) microparticles which are potent procoagulant factors that promote thrombin generation. Treatment with dabigatran etexilate alone prevented tumor-induced increases in circulating TF(+) microparticles and also decreased the numbers of tumor-induced activated platelets by 40%. These results show that co-treatment with dabigatran etexilate and CP synergistically inhibits growth and metastasis of mammary tumors, suggesting that oral administration of the thrombin inhibitor dabigatran etexilate may be beneficial in not only preventing thrombotic events in cancer patients but also in treating malignant tumors themselves.


Subject(s)
Antineoplastic Agents, Alkylating/pharmacology , Antithrombins/pharmacology , Cyclophosphamide/pharmacology , Neoplasms/pathology , Thrombin/antagonists & inhibitors , Animals , Cell Line, Tumor , Cell-Derived Microparticles/metabolism , Dabigatran/pharmacology , Disease Models, Animal , Disease Progression , Drug Synergism , Female , Mice , Myeloid Cells/drug effects , Myeloid Cells/immunology , Myeloid Cells/metabolism , Neoplasm Metastasis , Neoplasms/drug therapy , Neoplasms/immunology , Neoplasms/metabolism , Platelet Activation/drug effects , Thromboplastin/metabolism
9.
J Lipid Res ; 54(12): 3464-70, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24089247

ABSTRACT

It is important to understand HDL heterogeneity because various subspecies possess different functionalities. To understand the origins of HDL heterogeneity arising from the existence of particles containing only apoA-I (LpA-I) and particles containing both apoA-I and apoA-II (LpA-I+A-II), we compared the abilities of both proteins to promote ABCA1-mediated efflux of cholesterol from HepG2 cells and form nascent HDL particles. When added separately, exogenous apoA-I and apoA-II were equally effective in promoting cholesterol efflux, although the resultant LpA-I and LpA-II particles had different sizes. When apoA-I and apoA-II were mixed together at initial molar ratios ranging from 1:1 to 16:1 to generate nascent LpA-I+A-II HDL particles, the particle size distribution altered, and the two proteins were incorporated into the nascent HDL in proportion to their initial ratio. Both proteins formed nascent HDL particles with equal efficiency, and the relative amounts of apoA-I and apoA-II incorporation were driven by mass action. The ratio of lipid-free apoA-I and apoA-II available at the surface of ABCA1-expressing cells is a major factor in determining the contents of these proteins in nascent HDL. Manipulation of this ratio provides a means of altering the relative distribution of LpA-I and LpA-I+A-II HDL particles.


Subject(s)
ATP Binding Cassette Transporter 1/metabolism , Apolipoprotein A-II/pharmacology , Apolipoprotein A-I/pharmacology , Cholesterol, HDL/metabolism , Hep G2 Cells , Humans , Up-Regulation/drug effects
10.
Arterioscler Thromb Vasc Biol ; 33(4): 687-93, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23413428

ABSTRACT

OBJECTIVE: The goal of this study was to understand the molecular basis of how the amino acid substitution C112R that distinguishes human apolipoprotein (apo) E4 from apoE3 causes the more proatherogenic plasma lipoprotein-cholesterol distribution that is known to be associated with the expression of apoE4. APPROACH AND RESULTS: Adeno-associated viruses, serotype 8 (AAV8), were used to express different levels of human apoE3, apoE4, and several C-terminal truncation and internal deletion variants in C57BL/6 apoE-null mice, which exhibit marked dysbetalipoproteinemia. Plasma obtained from these mice 2 weeks after the AAV8 treatment was analyzed for cholesterol and triglyceride levels, as well as for the distribution of cholesterol between the lipoprotein fractions. Hepatic expression of apoE3 and apoE4 induced similar dose-dependent decreases in plasma cholesterol and triglyceride to the levels seen in control C57BL/6 mice. Importantly, at the same reduction in plasma total cholesterol, expression of apoE4 gave rise to higher very low-density lipoprotein-cholesterol (VLDL-C) and lower high-density lipoprotein-cholesterol levels relative to the apoE3 situation. The C-terminal domain and residues 261 to 272 in particular play a critical role, because deleting them markedly affected the performance of both isoforms. CONCLUSIONS: ApoE4 possesses enhanced lipid and VLDL-binding ability relative to apoE3, which gives rise to impaired lipolytic processing of VLDL in apoE4-expressing mice. These effects reduce VLDL remnant clearance from the plasma compartment and decrease the amount of VLDL surface components available for incorporation into the high-density lipoprotein pool, accounting for the more proatherogenic lipoprotein profile (higher VLDL-C/high-density lipoprotein-cholesterol ratio) occurring in apoE4-expressing animals compared with their apoE3 counterparts.


Subject(s)
Apolipoprotein E3/metabolism , Apolipoprotein E4/metabolism , Cholesterol, HDL/blood , Cholesterol, VLDL/blood , Cholesterol/blood , Hyperlipoproteinemia Type III/blood , Amino Acid Substitution , Animals , Apolipoprotein E3/chemistry , Apolipoprotein E3/deficiency , Apolipoprotein E3/genetics , Apolipoprotein E4/chemistry , Apolipoprotein E4/deficiency , Apolipoprotein E4/genetics , Dependovirus/genetics , Disease Models, Animal , Genetic Vectors , Humans , Hyperlipoproteinemia Type III/genetics , Lipolysis , Liver/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Protein Structure, Tertiary , Time Factors , Triglycerides/blood
11.
J Clin Invest ; 122(8): 2807-16, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22751103

ABSTRACT

Genome-wide association studies (GWAS) have identified a genetic variant at a locus on chromosome 1p13 that is associated with reduced risk of myocardial infarction, reduced plasma levels of LDL cholesterol (LDL-C), and markedly increased expression of the gene sortilin-1 (SORT1) in liver. Sortilin is a lysosomal sorting protein that binds ligands both in the Golgi apparatus and at the plasma membrane and traffics them to the lysosome. We previously reported that increased hepatic sortilin expression in mice reduced plasma LDL-C levels. Here we show that increased hepatic sortilin not only reduced hepatic apolipoprotein B (APOB) secretion, but also increased LDL catabolism, and that both effects were dependent on intact lysosomal targeting. Loss-of-function studies demonstrated that sortilin serves as a bona fide receptor for LDL in vivo in mice. Our data are consistent with a model in which increased hepatic sortilin binds intracellular APOB-containing particles in the Golgi apparatus as well as extracellular LDL at the plasma membrane and traffics them to the lysosome for degradation. We thus provide functional evidence that genetically increased hepatic sortilin expression both reduces hepatic APOB secretion and increases LDL catabolism, providing dual mechanisms for the very strong association between increased hepatic sortilin expression and reduced plasma LDL-C levels in humans.


Subject(s)
Adaptor Proteins, Vesicular Transport/metabolism , Apolipoproteins B/metabolism , Lipoproteins, LDL/metabolism , Liver/metabolism , APOBEC-1 Deaminase , Adaptor Proteins, Vesicular Transport/genetics , Animals , Apolipoproteins B/genetics , Base Sequence , Cell Line , Cytidine Deaminase/deficiency , Cytidine Deaminase/genetics , DNA Primers/genetics , Humans , Hydrogen-Ion Concentration , Kinetics , Lipoproteins, VLDL/metabolism , Lysosomes/metabolism , Mice , Mice, Knockout , Mice, Transgenic , Models, Biological , Mutagenesis, Site-Directed , Receptors, LDL/deficiency , Receptors, LDL/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
12.
Arterioscler Thromb Vasc Biol ; 31(2): 320-7, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21071688

ABSTRACT

OBJECTIVE: The goal of this study was to determine the influence of apolipoprotein A-I (apoA-I) tertiary structure domain properties on the antiatherogenic properties of the protein. Two chimeric hybrids with the N-terminal domains swapped (human-mouse apoA-I and mouse-human apoA-I) were expressed in apoA-I-null mice with adeno-associated virus (AAV) and used to study macrophage reverse cholesterol transport (RCT) in vivo. METHODS AND RESULTS: The different apoA-I variants were expressed in apoA-I-null mice that were injected with [H(3)]cholesterol-labeled J774 mouse macrophages to measure RCT. Significantly more cholesterol was removed from the macrophages and deposited in the feces via the RCT pathway in mice expressing mouse-H apoA-I compared with all other groups. Analysis of the individual components of the RCT pathway demonstrated that mouse-H apoA-I promoted ATP-binding cassette transporter A1-mediated cholesterol efflux more efficiently than all other variants, as well as increasing the rate of cholesterol uptake into liver cells. CONCLUSIONS: The structural domain properties of apoA-I affect the ability of the protein to mediate macrophage RCT. Replacement of the N-terminal helix bundle domain in the human apoA-I with the mouse apoA-I counterpart causes a gain of function with respect to macrophage RCT, suggesting that engineering some destabilization into the N-terminal helix bundle domain or increasing the hydrophobicity of the C-terminal domain of human apoA-I would enhance the antiatherogenic properties of the protein.


Subject(s)
Apolipoprotein A-I/chemistry , Apolipoprotein A-I/physiology , Cholesterol/metabolism , Macrophages/metabolism , ATP-Binding Cassette Transporters/metabolism , Adenoviridae/genetics , Animals , Apolipoprotein A-I/genetics , Biological Transport/genetics , Biological Transport/physiology , Humans , Macrophages/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Animal , Protein Structure, Tertiary/genetics , Protein Structure, Tertiary/physiology , Receptors, LDL/metabolism
13.
Arterioscler Thromb Vasc Biol ; 29(10): 1496-501, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19661486

ABSTRACT

OBJECTIVE: To compare the abilities of human wild-type apoA-I (WT apoA-I) and human apoA-I(Milano) (apoA-I(M)) to promote macrophage reverse cholesterol transport (RCT) in apoA-I-null mice infected with adeno-associated virus (AAV) expressing either WT apoA-I or apoA-I(M). METHODS AND RESULTS: WT apoA-I- or apoA-I(M)-expressing mice were intraperitoneally injected with [H(3)]cholesterol-labeled J774 mouse macrophages. After 48 hours, no significant difference was detected in the amount of cholesterol removed from the macrophages and deposited in the feces via the RCT pathway between the WT apoA-I and apoA-I(M) groups. Analysis of the individual components of the RCT pathway demonstrated that the apoA-I(M)-expressing mice promoted ATP-binding cassette transporter A1 (ABCA1)-mediated cholesterol efflux as efficiently as WT apoA-I but that apoA-I(M) had a reduced ability to promote cholesterol esterification via lecithin cholesterol-acyltransferase (LCAT). This resulted in reduced cholesteryl ester (CE) and increased free cholesterol (FC) levels in the plasma of mice expressing apoA-I(M) compared to WT apoA-I. These differences did not affect the rate of delivery of labeled cholesterol to the liver via SR-BI-mediated selective uptake or its subsequent excretion in the feces. CONCLUSIONS: Within the limits of the in vivo assay, WT apoA-I and apoA-I(M) are equally efficient at promoting macrophage RCT, suggesting that if apoA-I(M) is more atheroprotective than WT apoA-I it is not attributable to an enhancement of macrophage RCT.


Subject(s)
Apolipoprotein A-I/physiology , Cholesterol/metabolism , Macrophages/metabolism , ATP Binding Cassette Transporter 1 , ATP-Binding Cassette Transporters/physiology , Animals , Apolipoprotein A-I/genetics , Biological Transport , Cholesterol, HDL/blood , Mice , Mutation , Rats , Scavenger Receptors, Class B/physiology
14.
Arterioscler Thromb Vasc Biol ; 29(6): 837-42, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19325142

ABSTRACT

OBJECTIVE: The purpose of this study was to determine the effect of 15-lipoxygenase-1 (15-LO-1) on cholesterol mobilization from macrophages. METHODS AND RESULTS: Overexpression of human 15-LO-1 in RAW mouse macrophages led to enhanced cholesterol efflux, increased cholesteryl ester (CE) hydrolysis, and increased reverse cholesterol transport (RCT). Efflux studies comparing 15-LO-1 overexpressing cells to mock-transfected RAW macrophages resulted in a 3- to 7-fold increase in cholesterol efflux to apolipoprotein A-I and a modest increase in efflux to HDL. Additional experiments revealed an increase in mRNA and protein levels of ABCA1 and ABCG1 in the RAW expressing 15-LO-1 compared to controls. Efforts to examine whether the arachidonic acid metabolite of 15-LO-1, (15S)-hydroxyeicosatetraenoic acid (HETE), was responsible for the enhanced efflux revealed this eicosanoid metabolite did not play a role. Enhanced steryl ester hydrolysis was observed in 15-LO-1 overexpressing cells suggesting that the CE produced in the 15-LO-1 expressing cells was readily mobilized. To measure RCT, RAW macrophages overexpressing 15-LO-1 or mock-transfected cells were cholesterol enriched by exposure to acetylated low-density lipoprotein and [(3)H]-cholesterol. These macrophages were injected into wild-type animals and RCT was measured as a percent of injected dose of (3)H appearing in the feces at 48 hours. We found 7% of the injected (3)H in the feces of mice that received macrophages overexpressing 15-LO-1 and 4% in the feces of mice that received mock-transfected cells. CONCLUSIONS: These data are consistent with a model in which overexpression of human 15-LO-1 in RAW macrophages promotes RCT through increased CE hydrolysis and ABCA1-mediated cholesterol efflux.


Subject(s)
Arachidonate 15-Lipoxygenase/metabolism , Cholesterol Ester Transfer Proteins/metabolism , Cholesterol/metabolism , Macrophages/enzymology , ATP Binding Cassette Transporter 1 , ATP Binding Cassette Transporter, Subfamily G, Member 1 , ATP-Binding Cassette Transporters/metabolism , Animals , Apolipoprotein A-I/metabolism , Arachidonate 15-Lipoxygenase/genetics , Biological Transport , Cell Line , Cholesterol Esters/metabolism , Cholesterol, HDL/metabolism , Feces/chemistry , Humans , Hydrolysis , Hydroxyeicosatetraenoic Acids/metabolism , Lipoproteins/metabolism , Lipoproteins, LDL/metabolism , Macrophages/transplantation , Mice , RNA, Messenger/metabolism , Time Factors , Transfection , Tritium , Up-Regulation
15.
J Lipid Res ; 50(7): 1409-19, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19318685

ABSTRACT

Carriers of the apolipoprotein A-I(Milano) (apoA-I(M)) variant, R173C, have reduced levels of plasma HDL but no increase in cardiovascular disease. Despite intensive study, it is not clear whether the removal of the arginine or the introduction of the cysteine is responsible for this altered functionality. We investigated this question using two engineered variations of the apoA-I(M) mutation: R173S apoA-I, similar to apoA-I(M) but incapable of forming a disulfide bond, and R173K apoA-I, a conservative mutation. Characterization of the lipid-free proteins showed that the order of stability was wild type approximately R173K>R173S>R173C. Compared with wild-type apoA-I, apoA-I(M) had a lower affinity for lipids, while R173S apoA-I displayed intermediate affinity. The in vivo effects of the apoA-I variants were measured by injecting apoA-I-expressing adeno-associated virus into apoA-I-null mice. Mice that expressed the R173S variant again showed an intermediate phenotype. Thus, both the loss of the arginine and its replacement by a cysteine contribute to the altered properties of apoA-I(M). The arginine is potentially involved in an intrahelical salt bridge with E169 that is disrupted by the loss of the positively charged arginine and repelled by the cysteine, destabilizing the helix bundle domain in the apoA-I molecule and modifying its lipid binding characteristics.


Subject(s)
Apolipoprotein A-I , Mutation , Protein Structure, Secondary , Structure-Activity Relationship , Animals , Apolipoprotein A-I/chemistry , Apolipoprotein A-I/genetics , Apolipoprotein A-I/metabolism , Heterozygote , Humans , Lipid Metabolism , Lipoproteins, HDL/chemistry , Lipoproteins, HDL/metabolism , Lipoproteins, HDL/ultrastructure , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mutagenesis, Site-Directed , Particle Size , Protein Denaturation
16.
J Lipid Res ; 50 Suppl: S189-94, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19064999

ABSTRACT

Reverse cholesterol transport (RCT) is a term used to describe the efflux of excess cellular cholesterol from peripheral tissues and its return to the liver for excretion in the bile and ultimately the feces. It is believed to be a critical mechanism by which HDL exert a protective effect on the development of atherosclerosis. In this paradigm, cholesterol is effluxed from arterial macrophages to extracellular HDL-based acceptors through the action of transporters such as ABCA1 and ABCG1. After efflux to HDL, cholesterol may be esterified in the plasma by the enzyme lecithin:cholesterol acyltransferase and is ultimately transported from HDL to the liver, either directly via the scavenger receptor BI or after transfer to apolipoprotein B-containing lipoproteins by the cholesteryl ester transfer protein. Methods for assessing the integrated rate of macrophage RCT in animals have provided insights into the molecular regulation of the process and suggest that the dynamic rate of macrophage RCT is more strongly associated with atherosclerosis than the steady-state plasma concentration of HDL cholesterol. Promotion of macrophage RCT is a potential therapeutic approach to preventing or regressing atherosclerotic vascular disease, but robust measures of RCT in humans will be needed in order to confidently advance RCT-promoting therapies in clinical development.


Subject(s)
Atherosclerosis/metabolism , Cholesterol/metabolism , Animals , Biological Transport , Extracellular Space/metabolism , Humans , Liver/metabolism , Macrophages/metabolism
17.
Arterioscler Thromb Vasc Biol ; 27(9): 2022-9, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17615385

ABSTRACT

OBJECTIVE: The present study is a comparative investigation of cellular lipid mobilization and efflux to lipid-free human apoA-I and apoA-I(Milano), reconstituted high-density lipoprotein (rHDL) particles containing these proteins and serum isolated from mice expressing human apoA-I or apoA-I(Milano). METHODS AND RESULTS: Cholesterol and phospholipid efflux to these acceptors was measured in cell systems designed to assess the contributions of ATP-binding cassette A1 (ABCA1), scavenger receptor type BI (SRBI), and cellular lipid content to cholesterol and phospholipid efflux. Acceptors containing the Milano variant of apoA-I showed no functional increase in lipid efflux in all assays when compared with wild-type apoA-I. In fact, in some systems, acceptors containing the Milano variant of apoA-I promoted significantly less efflux than the acceptors containing wild-type apoA-I (apoA-I(wt)). Additionally, intracellular cholesteryl ester hydrolysis in macrophage foam cells was not different in the presence of either apoA-I(Milano) or apoA-I(wt). CONCLUSION: Collectively these studies suggest that if the Milano variant of apoA-I offers greater atheroprotection than wild-type apoA-I, it is not attributable to greater cellular lipid mobilization.


Subject(s)
Apolipoprotein A-I/physiology , Lipid Mobilization/physiology , ATP Binding Cassette Transporter 1 , ATP-Binding Cassette Transporters/metabolism , Animals , Cells, Cultured , Cholesterol Esters/metabolism , Humans , Lipoproteins, HDL2/metabolism , Macrophages, Peritoneal/physiology , Mice , Mice, Transgenic , Scavenger Receptors, Class B/metabolism
18.
J Biol Chem ; 280(38): 33015-25, 2005 Sep 23.
Article in English | MEDLINE | ID: mdl-15972827

ABSTRACT

The structure of apoA-I on discoidal high density lipoprotein (HDL) was studied using a combination of chemical cross-linking and mass spectrometry. Recombinant HDL particles containing 145 molecules of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and two molecules of apoA-I with a 96-A diameter were treated with the lysine-specific cross-linker, dithiobis(succinimidylpropionate) at varying molar ratios from 2:1 to 200:1. At low molar ratios of dithiobis(succinimidylpropionate) to apoA-I, two products were obtained corresponding to approximately 53 and approximately 80 kDa. At high molar ratios, these two products merged, yielding a product of approximately 59 kDa, close to the theoretical molecular mass of dimeric apoA-I. To identify the intermolecular cross-links giving rise to the two different sized products, bands were excised from the gel, digested with trypsin, and then analyzed by liquid chromatography-electrospray-tandem mass spectrometry. In addition, tandem mass spectrometry of unique cross-links found in the 53- and 80-kDa products suggested that a distinct conformation exists for lipid-bound apoA-I on 96-A recombinant HDL, emphasizing the inherent flexibility and malleability of the N termini and its interaction with its C-terminal domain.


Subject(s)
Apolipoprotein A-I/chemistry , Amino Acid Sequence , Chromatography, Liquid , Cross-Linking Reagents/pharmacology , Cysteine/chemistry , Dimerization , Electrophoresis, Polyacrylamide Gel , Glycerylphosphorylcholine/chemistry , Lipids/chemistry , Lysine/pharmacology , Mass Spectrometry , Models, Molecular , Molecular Sequence Data , Mutation , Oligonucleotides/chemistry , Peptides/chemistry , Protein Conformation , Protein Denaturation , Protein Folding , Protein Structure, Tertiary , Spectrometry, Mass, Electrospray Ionization , Succinimides/pharmacology , Trypsin/chemistry , Trypsin/pharmacology , Urea/pharmacology
19.
Biochemistry ; 44(14): 5409-19, 2005 Apr 12.
Article in English | MEDLINE | ID: mdl-15807534

ABSTRACT

Apolipoprotein A-I (apoA-I), the major protein in high density lipoprotein (HDL) regulates cholesterol homeostasis and is protective against atherosclerosis. An examination of the amino acid sequence of apoA-I among 21 species shows a high conservation of positively and negatively charged residues within helix 6, a domain responsible for regulating the rate of cholesterol esterification in plasma. These observations prompted an investigation to determine if charged residues in helix 6 maintain a structural conformation for protein-protein interaction with lecithin-cholesterol acyltransferase (LCAT) the enzyme for which apoA-I acts as a cofactor. Three apoA-I mutants were engineered; the first, (3)/(4) no negative apoA-I, eliminated 3 of the 4 negatively charged residues in helix 6, no negative apoA-I (NN apoA-I) eliminated all four negative charges, while all negative (AN apoA-I) doubled the negative charge. Reconstituted phospholipid-containing HDL (rHDL) of two discrete sizes and compositions were prepared and tested. Results showed that LCAT activation was largely influenced by both rHDL particle size and the net negative charge on helix 6. The 80 A diameter rHDL showed a 12-fold lower LCAT catalytic efficiency when compared to 96 A diameter rHDL, apparently resulting from an increased protein-protein interaction, at the expense of lipid-protein association on the 80 A rHDL. When mutant apoproteins were compared bound to the two different sized rHDL, a strong inverse correlation (r = 0.85) was found between LCAT catalytic efficiency and apoA-I helix 6 net negative charge. These results support the concept that highly conserved negatively charged residues in apoA-I helix 6 interact directly and attenuate LCAT activation, independent of the overall particle charge.


Subject(s)
Apolipoprotein A-I/metabolism , Phosphatidylcholine-Sterol O-Acyltransferase/metabolism , Apolipoprotein A-I/chemistry , Base Sequence , DNA Primers , Electrophoresis, Agar Gel , Kinetics , Particle Size , Phosphatidylcholine-Sterol O-Acyltransferase/chemistry
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