Labeled Low-density Lipoprotein Research Articles

Platelet Factor 4 Binds to Low-Density Lipoprotein Receptor to Block Leukemic Stem Cell Expansion and Recurrence

Healthy hematopoietic stem cells (HSCs) reside in highly specialized microenvironments within the bone marrow, commonly referred to as niches. HSC activity is tightly regulated by various cellular and molecular niche constituents that ensure healthy hematopoiesis. However, through the accumulation of mutations, hematopoietic stem and progenitor cells can give rise to leukemia-initiating cells or leukemic stem cells (LSCs), which play a vital role in the onset, progression, and treatment outcome of acute myelogenous leukemia (AML). In AML, the expansion of the leukemic clone is associated with impaired healthy hematopoiesis, resulting in severe anemia, thrombocytopenia, and immunodeficiency, which can lead to severe morbidity. Current therapeutic options mainly eliminate the bulk of leukemic cells but do not efficiently target LSCs, leading to the high rates of relapse seen in AML patients. Recent studies suggest that LSCs hijack and alter the healthy bone marrow niche to better support their maintenance and proliferation. Understanding how LSCs interact with the bone marrow microenvironment is crucial for eradicating LSCs and preventing AML relapse. Our previous work showed that platelet factor 4 (PF4), a chemokine stored in platelet alpha-granules and secreted by bone marrow megakaryocytes, can induce quiescence in healthy myeloid-biased HSCs ( Nature Medicine 2014; Developmental Cell 2018). Here, we investigate the regulation of malignant LSCs by PF4 using mouse and human AML models. In an aggressive mouse AML model, MLL-AF9, our results show that recombinant PF4 blocks the proliferation of phenotypic LSCs (Lineage − IL7Rα − Sca1 − MLL-AF9-GFP + c-Kit high CD34 + FcγRII/III high) in vitro (70% reduction; P=0.0279). Strikingly, cell cycle analyses revealed that in vivo, both LSCs and bulk AML proliferation are significantly inhibited 24 hours post-PF4 (80 mg/kg) injection, resulting in a significantly lower leukemic burden. By contrast, Pf4 -/- mice exhibited rapid AML disease progression via increased LSC proliferation. Furthermore, using a genetic mouse model ( Pf4-Cre; iDTR) where PF4-secreting megakaryocyte cells can be depleted in vivo in leukemic mice, our results revealed that megakaryocyte depletion led to poor AML survival due to accelerated disease progression (Log-rank P<0.0017). These results demonstrate that megakaryocyte-derived PF4 can directly regulate the proliferation of leukemia cells. Interestingly, our results indicate that AML drives striking alterations in bone marrow megakaryocytes, including a lower frequency, reduced cell maturation, and reduced secretion of PF4 (~80% reduction, P<0.0001) compared to healthy controls. Accordingly, PF4 levels are reduced in the serum of myelodysplastic syndrome patients where hyperproliferative malignant cells are found alongside dysplastic megakaryocytes. To assess whether recombinant PF4 can alter the course of AML relapse in a preclinical experimental setting, we treated moribund leukemic mice with the chemotherapeutic drug Cytarabine followed by 3 daily I.V. injections of PF4 (80 mg/kg) or saline. Strikingly, this short treatment with PF4 preferentially reduced the frequency of LSCs in vivo during the relapse phase of AML (75% reduction; P=0.05). Furthermore, mice transplanted with MLL-AF9 AML cells that were pre-treated with Cytarabine and PF4 in vitro had prolonged overall survival (Log-rank P<0.0132) compared to Cytarabine alone. Importantly, two of six mice had no detectable AML up to 150 days post-transplantation. Finally, single-cell RNA sequencing experiments revealed that PF4 signals through Low-Density Lipoprotein Receptor (LDLR) on LSCs, whose expression is significantly upregulated upon Cytarabine treatment, during the relapse phase. Functional studies with fluorescently labeled low-density lipoprotein (LDL) confirmed that PF4 limits LSC uptake of LDL via LDLR to block LSC proliferation ( P=0.0044), suggesting an important role for PF4 in AML metabolism. Altogether, our studies reveal an unknown function for PF4 in AML and highlight PF4's potential as an effective adjuvant therapy in preventing AML relapse by inhibiting the recurrence of chemotherapeutic-resistant LSCs.

Abstract 2881: Investigating cholesterol transfer between macrophages and prostate cancer cells

Abstract Background: Androgens stimulate androgen receptor (AR) signaling, driving prostate cancer (PC) cell proliferation and progression. Androgen deprivation therapy (ADT) effectively treats primary disease, but 20-30% of patients relapse after ~5 years, resulting in castration-resistant prostate cancer (CRPC), which has limited treatment options. We have previously shown in a murine model of CRPC that macrophages promote AR signaling in CRPC and that depleting tumor macrophages extends survival in response to ADT with Lupron. This correlates with reduced androgen levels in macrophage-depleted tumors and the ability of macrophages to transfer cholesterol, the anabolic precursor of androgens, to prostate cancer cells. However, the mechanism by which this transfer occurs, and whether cholesterol exchange mediates resistance to ADT, is currently unknown. Methods: To assess cholesterol transfer from macrophages to cancer cells, we established a culture system involving prostate cancer cell lines and either the RAW264.7 macrophage cell line or bone marrow-derived macrophages (BMDMs). Macrophages were pre-loaded with cholesterol using fluorescently labeled low-density lipoprotein (LDL) and were co-cultured or placed in a transwell system with tumor cells for 24 hours. Cholesterol transfer was then measured by flow cytometry. Results: Fluorescent signal from LDL was detected in 50-70% of tumor cells after 24 hours of co-culture. Cell contact was required for cholesterol transfer, as no fluorescence was detected in prostate cancer cells during transwell experiments. Macrophages were also able to internalize and transfer acetylated LDL, which is poorly taken up by tumor cells compared to unmodified LDL. Macrophage polarization with interleukin-4 did not impact cholesterol transfer and uptake of acetylated LDL occurred independently of the scavenger receptor CD36. The scavenger receptor SR-B1, which is often upregulated in prostate cancer cells, was dispensable for transfer, as knocking out SR-B1 via CRISPR/Cas9 in cancer cell lines did not reduce LDL uptake. Conclusion: Cholesterol transfer between macrophages and prostate cancer cells occurs in a contact-dependent manner, independently of the scavenger receptors most commonly associated with cholesterol accumulation. Citation Format: Olabisi Osunmakinde, Brian Ruffell. Investigating cholesterol transfer between macrophages and prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2881.

Infection of Hepatocytes With HCV Increases Cell Surface Levels of Heparan Sulfate Proteoglycans, Uptake of Cholesterol and Lipoprotein, and Virus Entry by Up-regulating SMAD6 and SMAD7

The signaling molecule and transcriptional regulator SMAD6, which inhibits the transforming growth factor β signaling pathway, is required for infection of hepatocytes by hepatitis C virus (HCV). We investigated the mechanisms by which SMAD6 and another inhibitory SMAD (SMAD7) promote HCV infection in human hepatoma cells and hepatocytes. We infected Huh7 and Huh7.5.1 cells and primary human hepatocytes with Japanese fulminant hepatitis-1 (JFH1) HCV cell culture system (HCVcc). We then measured HCV binding, intracellular levels of HCV RNA, and expression of target genes. We examined HCV entry in HepG2/microRNA (miR) 122/CD81 cells, which support entry and replication of HCV, were transfected these cells with small interfering RNAs targeting inhibitory SMADs to analyze gene expression profiles. Uptake of labeled low-density lipoprotein (LDL) and cholesterol was measured. Cell surface proteins were quantified by flow cytometry. We obtained liver biopsy samples from 69 patients with chronic HCV infection and 19 uninfected individuals (controls) and measured levels of syndecan 1 (SDC1), SMAD7, and SMAD6 messenger RNAs (mRNAs). Small interfering RNA knockdown of SMAD6 blocked the binding and infection of hepatoma cell lines and primary human hepatocytes by HCV, whereas SMAD6 overexpression increased HCV infection. We found levels of mRNAs encoding heparan sulfate proteoglycans (HSPGs), particularly SDC1 mRNA, and cell surface levels of heparan sulfate to be reduced in cells after SMAD6 knockdown. SMAD6 knockdown also reduced transcription of genes encoding lipoprotein and cholesterol uptake receptors, including the LDL receptor (LDLR), the very LDLR, and the scavenger receptor class B member 1 in hepatocytes; knockdown of SMAD6 also inhibited cell uptake of cholesterol and lipoprotein. Overexpression of SMAD6 increased the expression of these genes. Similar effects were observed with knockdown and overexpression of SMAD7. In addition, HCV infection of cells increased the expression of SMAD6, which required the activity of nuclear factor-κB, but not transforming growth factor β. Liver tissues from patients with chronic HCV infection had significantly higher levels of SMAD6, SMAD7, and HSPG mRNAs than controls. In studies of hepatoma cell lines and primary human hepatocytes, we found that infection with HCV leads to activation of nuclear factor-κB, resulting in increased expression of SMAD6 and SMAD7. Up-regulation of SMAD6 and SMAD7 induces the expression of HSPGs, such as SDC1, as well as LDLR, very LDLR, and the scavenger receptor class B member 1, which promote HCV entry and propagation, as well as cellular uptake of cholesterol and lipoprotein.

Disease-Corrected Hepatocyte-Like Cells from Familial Hypercholesterolemia-Induced Pluripotent Stem Cells

The generation of human induced pluripotent stem cells (hiPSCs) from an individual patient provides a unique tool for disease modeling, drug discovery, and cell replacement therapies. Patient-specific pluripotent stem cells can be expanded in vitro and are thus suitable for genetic manipulations. To date, several genetic liver disorders have been modeled using patient-specific hiPSCs. Here, we present the generation of corrected hepatocyte-like cells (HLCs) from hiPSCs of a familial hypercholesterolemia (FH) patient with a homozygous mutation in the low-density lipoprotein receptor (LDLR) gene. We generated hiPSCs from a patient with FH with the mutated gene encoding a truncated non-functional receptor. In order to deliver normal LDLR to the defective cells, we used a plasmid vector carrying the normal receptor ORF to genetically transform the hiPSCs. The transformed cells were expanded and directed toward HLCs. Undifferentiated defective hiPSCs and HLCs differentiated from the defective hiPSCs did not have the ability to uptake labeled low-density lipoprotein (LDL) particles. The differentiated transformed hiPSCs showed LDL-uptake ability and the correction of disease phenotype as well as expressions of hepatocyte-specific markers. The functionality of differentiated cells was also confirmed by indo-cyanine green (ICG) uptake assay, PAS staining, inducible cyp450 activity, and oil red staining. These data suggest that hiPSC technology can be used for generation of disease-corrected, patient-specific HLCs with potential value for disease modeling and drug discovery as well as cell therapy applications in future.

Endothelial Blood-Bone Marrow-Barrier Dynamically Regulates Balanced Stem and Progenitor Cell Trafficking and Maintenance

AbstractAbstract 507Bone marrow (BM) endothelial cells (BMECs) serve as ‘niche' cells for both hematopoietic and mesenchymal stem and progenitor cells (HSPC/MSPC). Yet BMECs control of HSPC bi-directional trafficking between the BM and peripheral blood (PB) through the blood-bone marrow-barrier (BBMB) is poorly understood.In vivo treatment with the pro-angiogenic cytokine FGF-2 reduced BM CXCL12 levels, and functionally upregulated CXCR4 expression on primitive Lineage−/Sca-1+/c-Kit+ (LSK) HSPCs accompanied by their increased in vitro CXCL12-induced migration. However, instead of expected HSPC mobilization effect, FGF-2 treatment resulted in reduced numbers of HSPCs in the PB while increasing BM HSPC levels. Based on this, we hypothesized that FGF-2 modifies the BM vascular endothelium, which may be responsible for the observed increase in HSPC retention. Accordingly, we examined BBMB permeability by injecting fluorescently labeled Low Density Lipoprotein (LDL). FGF-2 treatment resulted in reduced BM penetration and incorporation of LDL. Moreover, BM homing of HSPCs was significantly reduced in FGF-2 treated recipients. To further examine endothelial involvement, applying the murine CRE-Lox system for conditional gene knock-out, we used VE-Cadherin-CREERT2 transgenic mice crossed with FGFR1flox/flox/FGFR2flox/flox mice to generate endothelial specific tamoxifen-inducible knock-out of the two endothelial predominantly expressed FGF receptors (eFGFR1/2 KO). FGF-2 treatment of eFGFR1/2 KO mice did not exhibit increased BM retention and reduced homing capabilities of HSPCs as in WT treated mice, suggesting that endothelial specific activation of FGF signaling regulates the BBMB functional control of HSPC bi-directional trafficking. Importantly, eFGFR1/2 KO mice exhibited reduced levels of CD45−/CD11b−/Ter-119−/Sca-1low/-/PDGFβR+ MSPCs together with reduced levels of CD34−LSK and SLAM HSPCs. FGF-2 treatment increased both HSPC and MSPC levels in WT, but not in eFGFR1/2 KO mice. These results imply that hampering endothelial FGF signaling interferes with HSPC/MSPC maintenance, while increasing BBMB permeability. Mechanistically, FGF-2 treatment resulted in decreased MMP-9 protease activity levels in BM supernatants combined with upregulated Timp-1 (an endogenous MMP-9 inhibitor) mRNA levels in total BM cells. Furthermore, FGF-2 treatment reduced eNOS phosphorylation and NO content in total BM cells and in BMECs. As both NO and MMP-9 can promote VE-Cadherin shedding, and subsequently increase endothelial barrier permeability, we observed increased VE-Cadherin expression levels on BMECs following FGF-2 treatment. Supporting this notion, in vivo administration of neutralizing VE-Cadherin antibodies efficiently increased HSPC egress by increasing BBMB permeability. Additionally, VE-Cadherin neutralization increased HSPC BM homing and LDL incorporation. These results reveal that interfering with endothelial adhesion interactions increases HSPC egress and homing. Examination of eNOS KO, Timp-1 KO and WT mice revealed that during steady state, mature WBC counts in the PB were similar. However, PB HSPC numbers were decreased in eNOS KO mice and increased in Timp-1 KO mice as measured by CFU-C and LSK. Similarly, HSPC BM homing capacity and LDL incorporation were decreased in eNOS KO mice and increased in Timp-1 KO mice. These results suggest that NO and MMP-9 mediated shedding of VE-Cadherin promotes BBMB permeability, which regulates egress and homing of immature HSPCs.In conclusion, our findings reveal that FGF-2-induced expansion of both HSPCs and MSPCs involves BMEC activation. Yet, FGF-stimulated HSPCs fail to egress into the PB and are retained in the BM due to decreased endothelial BBMB permeability. Thus, FGF signaling in BMECs may serve as a “gate-keeper” for HSPC trafficking synchronized with HSPC/MSPC maintenance. We suggest that BMECs are dynamically balanced between their dual role as a ‘niche', regulating HSPC and MSPC maintenance and as a selective, anatomical barrier regulating HSPC bi-directional trafficking. When the BBMB restricts HSPC trafficking via reduced permeability, it provides better HSPC/MSPC support and maintenance. On the other hand, upon functioning as a trafficking site with increased BBMB permeability, BMEC-provided support and maintenance is reduced. Disclosures:No relevant conflicts of interest to declare.

Cytoskeletal changes during epithelial-to-fibroblastoid conversion as a crucial mechanism of des-γ-carboxy prothrombin production in hepatocellular carcinoma

Des-gamma-carboxy prothrombin (DCP) is a well-established tumor marker for hepatocellular carcinoma (HCC), but the precise mechanism by which HCC cells produce DCP remains unknown. Our preliminary experiments demonstrated that HepG2 cells with chemical induction of epithelial-to-fibroblastoid conversion (EFC) produced DCP through impairment of vitamin K uptake via cytoskeletal rearrangement. Therefore, in this study we further examined this mechanism in vitro and using human HCC samples. Hepatoma cell lines (HepG2 and PLC/PRF/5) were induced EFC or epithelial-mesenchymal transition (EMT) by phorbol 12-myristate 13 acetate (TPA) or transforming growth factor (TGF)-beta1. We analyzed these cells by ELISA, Western blotting and immunofluorescent studies. We also examined DCP production and E-cadherin expression in human surgically resected HCC samples by immunohistochemical studies. Labeled low-density lipoprotein (LDL) uptake (as a surrogate for vitamin K) was significantly impaired in DCP-producing hepatoma cells following induction of EFC or EMT. Further, filamentous actin, which plays a critical role in clathrin-mediated endocytosis, was dissociated in DCP-producing cells. Latrunculin A, an actin depolymerizer, induced naïve hepatoma cells to produce DCP with impairment of labeled-LDL uptake. In addition, an E-cadherin neutralizing antibody did not induce DCP production. Finally, immunohistochemical studies demonstrated that DCP production was inversely correlated with the intensity of E-cadherin expression in human HCC cells. In conclusion, cytoskeletal changes during EFC or EMT plays a critical mechanistic role in DCP production via impairments in vitamin K uptake.

Development of a lipoprotein based molecular imaging MR contrast agent for the noninvasive detection of early atherosclerotic disease

Currently there are no clinically available means of noninvasively detecting early atherosclerotic disease because these lesions are characterized by an accumulation of extracellular lipid and foam cells, but a lack of significant wall thickening or architectural distortion. We hypothesize that a paramagnetically labeled low density lipoprotein (LDL) could serve as a functional probe to detect sites of abnormal lipid metabolism in the vessel wall that represent sites of early disease. Isolated LDL was first incubated with manganese-mesoporphyrin, a hydrophobic MR contrast agent (MnMeso). Size exclusion chromatography and absorption mass spectroscopy were performed on the resulting samples to prove that an association between the two occurred. Subsequently, foam cell cultures (n=7) were incubated (10-30 microg/ml for 48 h) with these labeled lipoproteins and the T1 relaxivity of centrifuged pellets of these cells was determined by using an inversion recovery sequence on a 1.5T scanner. These results were compared to control measurements made from foam cell cultures fed unlabeled lipoproteins (n=7). Measured T1 relaxation times of the cells fed the MnMeso-LDL (443.3 +/- 51.8 ms) was significantly different from the T1 relaxivity obtained from cells fed unlabeled lipoproteins (661.3 +/- 60.9 ms). These findings indicate that the amount of contrast bound to the constructed lipoproteins is sufficient to produce measurable MR signal changes noninvasively. The study results support the feasibility of future in vivo MR experiments with labeled lipoproteins to assess lipoprotein kinetics in the vessel wall, which will hopefully provide a means of detecting early atherosclerotic disease.

Transfer of Phosphatidylethanol Between Lipoproteins

Phosphatidylethanol (PEth) is an abnormal phospholipid formed only in the presence of ethanol. It has been recently shown that lipoprotein-associated PEth may mediate the effects of ethanol on endothelial cells, and this may explain, at least in part, the beneficial effect of ethanol on atherosclerosis. This study was performed to investigate the transfer of PEth between lipoproteins and the effects of PEth on cholesteryl ester transfer protein (CETP) activity in plasma. Lipoproteins were isolated from the plasma of healthy male volunteers (n = 16) and male alcoholics (n = 13). The transfer of cholesteryl esters and PEth was determined between labeled low-density lipoprotein (LDL) and unlabeled high-density lipoprotein particles in vitro. The electrophoretic mobility of PEth-modified LDL particles was determined by agarose gel electrophoresis. PEth was transferred from PEth-modified LDL to high-density lipoprotein at an initial rate of 25.9 nmol/ml/hr. Monoclonal antibody (TP2) against the putative lipid-binding domain of CETP inhibited the transfer rate of PEth by approximately 64%, whereas the cholesteryl ester transfer was inhibited by 86%. This indicates that most of PEth was transferred by transfer proteins other than CETP. The transfer of PEth between lipoproteins enables the redistribution of PEth from lipoprotein fractions with a slow turnover to those with a rapid clearance. Moreover, the PEth-induced change in the electrical charge of lipoproteins may affect the binding of lipoproteins to their receptors and binding proteins. This in turn may alter the metabolism of lipoproteins and lipid-mediated signaling pathways in the cells delineating the vascular wall.

Carbocyanine labeled LDL for optical imaging of tumors 1

Rationale and objectives The purpose of this study was to define and characterize carbocyanine labeled low-density lipoprotein (LDL) to be used in the optical imaging of LDL receptor (LDLr)-overexpressing tumor models. Materials and methods 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) was used to label LDL (DiI-LDL). Scatchard plots were generated to determine the maximum binding capacity B max and dissociation constants K D of DiI-LDL in B16 melanoma (B16) and hepatoblastoma G 2 (HepG 2) cell lines. Selective uptake of DiI-LDL into both tumor cells and corresponding subcutaneous tumors in mice were demonstrated by confocal microscopy and three-dimensional Cryo-imaging, respectively. Results The labeling efficiency of DiI-LDL was 61 ng DiI/μg LDL protein (34 mol DiI/mol LDL protein). B max and K D for B16 cells were 6.311 ng LDL/mg cell protein and 60.38 μg protein/mL (117 nM), respectively. B max and K D were 7.573 ng LDL/mg cell protein and 26.79 μg protein/mL (52 nM) for HepG 2 cells, respectively. Confocal microscopic images showed specific uptake of DiI-LDL throughout the cytoplasm in the B16/HepG 2 cells. Cryo-imaging demonstrated preferential accumulations of DiI-LDL in the viable tumor regions of both B16 and HepG 2 tumors compared with their adjacent normal tissues and corresponding necrotic tumor regions. In addition, uptake of DiI-LDL by the HepG 2 tumor was much higher than that of the B16 tumor, consistent with the fact that the probe binding affinity for LDLrs of HepG 2 cells is 2.3 times that of B16 cells. Conclusion This study suggested that carbocyanine labeled LDL could be used for optical imaging of tumors overexpressing LDLr.

Melatonin Inhibits LDL Receptor Activity and Cholesterol-Synthesis in Freshly Isolated Human Mononuclear Leukocytes

There is some indirect evidence that the pineal hormone melatonin can suppress plasma levels of cholesterol in hypercholesterolemic rats. We have examined the effects of the hormone on cellular cholesterol metabolism in freshly isolated human mononuclear leukocytes. Incubation of cells for up to 20 h in a lipid-free medium resulted in an increase in the rate of cholesterol synthesis from [14C]acetate and the high affinity accumulation and degradation of [125I]labeled low density lipoprotein (LDL). Addition of melatonin in increasing concentrations to the incubation medium at zero time inhibited cholesterol synthesis and the specific accumulation and degradation of [125I]labeled LDL; at a concentration of 100 μM, the inhibitions were 38 %, 42 %, and 48 %, respectively. Similar results were obtained using [14C]mevalonate as precursor. Fatty acid synthesis was not altered under these conditions. In contrast to cholesterol the synthesis of the first cyclic compound lanosterol was not affected by the pineal hormone. These results implicate that melatonin inhibits this pathway between lanosterol and cholesterol. The action of melatonin on LDL receptor activity appeared to be mediated by a decrease in the number of LDL receptors and not by a change in binding affinity. Pharmacological characterization of the potential melatonin receptor site using several analogs hire tryptamine, 5-hydroxytryptamine, N-acetyl-5-hydroxtryptamine, 5-methoxytryptamine, and 6-chloromelatonin indicated that the 5-methoxy group is indispensible for the hormone action on cholesterol synthesis. The data provide evidence that melatonin can modulate cholesterol metabolism in human cells.

Optimal design of experiments to estimate LDL transport parameters in arterial wall

To quantify transport processes in atherosclerosis, the arterial wall is often exposed to labeled lipoproteins. In vivo experiments are desirable for estimation of transport parameters, but they are technically difficult. A dynamic mass transfer model has been developed to describe experimental transmural profiles of lipoprotein accumulation as a function of luminal permeability, diffusion, convection, and degradation. To avoid extraneous experiments and to assure successful parameter estimation, an optimal design of experiments is needed. For our purposes a design was considered optimal when it maximized the sensitivity of the model output to changes in parameter values as indicated by the determinant of the Hessian matrix of the objective function. A comparison was made between two designs: dual-time designs prescribing unequal circulation times for two distinguishable injections of labeled low-density lipoprotein (LDL) and dual-species designs requiring simultaneous circulation of LDL and tyramine-cellobiose-modified LDL. Circulation time was optimized for both designs. Although both were heavily dependent on the circulation times, dual-time designs required better preliminary knowledge of parameter values. Because labeled degradation products of the modified tracer become anchored in the arterial tissue, information about the degradation process is retained in the dual-species study. For this reason, dual-species designs were generally superior to dual-time designs.

Fluorescence studies of macrophage recognition and endocytosis of native and acetylated low-density lipoprotein

Macrophage recognition and endocytosis of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (diI)-labeled low-density lipoprotein (LDL) and acetyl LDL (Ac-LDL) was studied using fluorescence flow cytometry and fluorescence video intensification microscopy. RAW264 macrophages and U937 monocytes were grown in the tissue culture media in the presence and absence of LDL and Ac-LDL. Several lines of evidence indicate that receptor-mediated endocytosis of diI-labeled LDL or Ac-LDL was taking place. Binding can be distinguished from binding plus endocytosis by incubation at 4 and 37°C, respectively. Binding was saturable at 4°C and uptake at 37°C was time- and ligand dose-dependent. Also, unlabeled LDL and Ac-LDL compete for their receptors. Macrophages grown in the presence or absence of LDL demonstrated distinct labeling patterns. LDL receptors were significantly increased by culture in defined medium without serum lipoproteins, while Ac-LDL receptors remained unaffected. Flow cytometry can provide an important tool to examine receptor levels, modulation of these levels and receptor-mediated endocytosis. Video intensification microscopy of similarly labeled cells has been performed. Receptors appear as punctate fluorescence, usually distributed randomly across the cell surface.

REGULATORY ROLE OF TRIIODOTHYRONINE IN THE DEGRADATION OF LOW DENSITY LIPOPROTEIN BY CULTURED HUMAN SKIN FIBROBLASTS

Degradation of [125I]-labeled low density lipoprotein (LDL) by cultured normal human skin fibroblasts was enhanced by exposure to concentrations of L-triiodothyronine (T3) spanning the physiological range. Analysis of LDL concentration curves suggests that T3 may increase LDL receptor number rather than alter the affinity of LDL for its receptor. No effect of T3 on LDL degradation was observed in receptor negative cells. Thus thyroid hormone appears to regulate receptor-mediated LDL degradation by increasing the binding of the lipoprotein to its cell-surface receptor.

Endogenously labeled low density lipoprotein triglyceride and apoprotein B kinetics.

The kinetics of endogenously labeled low density lipoprotein (LDL) triglycerides (TG) and apoprotein B (apoB) have been studied in four normal and in four hyperlipemic subjects using double tracers. Analysis of the data suggests that most LDL triglycerides turn over about 10 times faster than apoB (0.003/min vs. 0.0003/min) and that about 10% of the LDL particles contain most of the TG found with LDL. It is not possible to determine from the analysis whether each new LDL particle arrives with the excess TG or whether only a subpopulation of particles contains most of the TG. The kinetic analysis further suggests that triglyceride-rich LDL particles do not exchange with an extraplasma compartment as most LDL particles do, and thus, they behave more like very low density lipoprotein particles. A compartmental model accounting for both the LDL-TG and LDL-apoB kinetics is proposed.