Yutaka Nagano

Fractalkine in vascular biology : from basic research to clinical disease

Abstract—Fractalkine (now also called CX3CL1) is a unique chemokine that functions not only as a chemoattractant but also as an adhesion molecule and is expressed on endothelial cells activated by proinflammatory cytokines, such as interferon-&ggr; and tumor necrosis factor-&agr;. The fractalkine receptor, CX3CR1, is expressed on cytotoxic effector lymphocytes, including natural killer (NK) cells and cytotoxic T lymphocytes, which contain high levels of intracellular perforin and granzyme B, and on macrophages. Soluble fractalkine causes migration of NK cells, cytotoxic T lymphocytes, and macrophages, whereas the membrane-bound form captures and enhances the subsequent migration of these cells in response to secondary stimulation with other chemokines. Furthermore, stimulation through membrane-bound fractalkine activates NK cells, leading to increased cytotoxicity and interferon-&ggr; production. Recently, accumulating evidence has shown that fractalkine is involved in the pathogenesis of various clinical disease states or processes, such as atherosclerosis, glomerulonephritis, cardiac allograft rejection, and rheumatoid arthritis. In addition, polymorphisms in CX3CR1, which reduce its binding activity to fractalkine, have been reported to increase the risk of HIV disease and to reduce the risk of coronary artery disease. This review will examine new concepts underlying fractalkine-mediated leukocyte migration and tissue damage, focusing primarily on the pathophysiological roles of fractalkine in various clinical conditions, especially in atherosclerosis and vascular injury.

Fractalkine, a CX 3 C-chemokine, functions predominantly as an adhesion molecule in monocytic cell line THP-1

A newly identified CX3C‐chemokine, fractalkine, expressed on activated endothelial cells plays an important role in leucocyte adhesion and migration. Co‐immobilized fractalkine with fibronectin or intercellular adhesion molecule‐1 enhanced adhesion of THP‐1 cells, which express the fractalkine receptor (CX3CR1), compared with that observed for each alone. That adherence was fractalkine‐dependent and was confirmed in blocking studies. However, soluble fractalkine induced little chemotaxis in THP‐1 cells in comparison to monocyte chemotactic protein‐1 (MCP‐1), which induced a strong chemotactic response. Moreover, the membrane form of fractalkine expressed on ECV304 cells reduced MCP‐1 mediated chemotaxis of THP‐1 cells. These results indicate that fractalkine may function as an adhesion molecule between monocytes and endothelial cells rather than as a chemotactic factor.

Probucol and atherosclerosis in the Watanabe heritable hyperlipidemic rabbit — long-term antiatherogenic effect and effects on established plaques

We performed two studies to investigate the effect of probucol on atherogenesis in vivo in the Watanabe heritable hyperlipidemic (WHHL) rabbit. In the first study (Study A), probucol was administered to 2-month-old WHHL rabbits, to evaluate its long-term effect. When killed at about 1.5 years of age, the percentage area of aorta covered with atherosclerotic plaque in probucol-treated rabbits was markedly less than that seen in non-treated rabbits (23.0 +/- 11.4% vs. 87.7 +/- 8.1%, M +/- S.D., P less than 0.001). In the second study (study B), administration of probucol was commenced with 8-month-old WHHL rabbits to investigate whether the drug was effective for limiting atherosclerosis in rabbits in which plaques had already developed. When killed after 6 months of treatment, the percentage area of aorta covered with plaque was 38.1 +/- 12.1% in treated rabbits and 82.7 +/- 22.6% in non-treated rabbits (P less than 0.02). Microscopic observations of lesions also supported the effect of probucol. Probucol treatment resulted in a change not only in the size but also the composition of lesions. Thus, probucol was effective in preventing atherosclerosis in long-term studies at both early and late stages.

Prevention of atherosclerotic progression in Watanabe rabbits by probucol

The foam cell has been recognized as a characteristic feature of xanthomas in skin and tendons, and also of atheromas. Many foam cells in these lesions share properties characteristic of the macrophages. Therefore macrophages may be the progenitor of certain foam cells that are involved in atherogenesis. Several investigators demonstrated in vitro that macrophages can ingest large amounts of certain chemically modified lipoproteins, such as acetylated low-density lipoprotein (LDL) and malondialdehyde-treated LDL, through the process of receptor-mediated endocytosis. By this process, macrophages become foam cells. But this process has not been demonstrated in vivo. Recently, oxidized LDL has been suggested to play an important role in atherogenesis by facilitating the accumulation of lipids in macrophages in vitro. Probucol, originally developed as an antioxidant, prevents this oxidative modification of LDL in vitro. Moreover, there are some clinical reports that probucol induces regression of cutaneous and tendon xanthomas in patients with homozygous familial hypercholesterolemia. A question was posed whether in vivo probucol could prevent the progression of atherosclerosis in homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits, an animal model for familial hypercholesterolemia. At age 2 months, 8 WHHL rabbits were classified into 2 groups: group A rabbits were controls and group B rabbits were treated with 1% probucol. After 6 months of treatment, average plasma concentrations of cholesterol were 704 +/- 121 mg/dl in group A and 584 +/- 61 mg/dl in group B. The percentage of surface area of total thoracic aorta with visible plaques in group A vs group B was 54.2 +/- 18.8% vs 7.0 +/- 6.3%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholesterol efflux effect of high density lipoprotein is impaired by whole cigarette smoke extracts through lipid peroxidation.

It has been reported that high density lipoprotein (HDL) plays an anti-atherogenic role by stimulating cholesterol efflux from the foam cells in the atheromatous lesion. In this study, we prepared a novel modified form of HDL (CS-HDL) by incubating HDL with whole cigarette smoke (CS) extracts containing both particulate matter and gas-phase smoke, and examined its effect on cholesterol efflux. CS-HDL showed a marked increase of conjugated dienes and denaturation of apoA-I, a major protein component of HDL. The cholesterol efflux effect of CS-HDL was remarkably reduced to the same level as that of oxidatively modified HDL induced by copper ion (Ox-HDL). Addition of 20 microg/ml superoxide dismutase (SOD) during the CS-modification of HDL caused retrieval of cholesterol efflux activity by 53% and a remarkable decrease in the conjugated dienes level. SOD, however, had no ameliorative effect on apoA-I denaturation. When HDL was incubated only with gas-phase smoke (gasCS-HDL), neither increase of conjugated dienes nor impairment of the cholesterol efflux effect was observed, whereas apoA-I was denaturated to the same extent as seen in CS-HDL. These results indicate that whole CS-extracts, but not gas-phase smoke, reduces cholesterol efflux effect of HDL and that lipid peroxidation associated with superoxide anion is involved in this functional impairment.

Induction of mRNA for low-density lipoprotein receptors in heterozygous Watanabe heritable hyperlipidemic rabbits treated with CS-514 (pravastatin) and cholestyramine

We administered CS-514, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, alone and in combination with cholestyramine to heterozygous Watanabe heritable hyperlipidemic rabbits. This rabbit model for heterozygous familial hypercholesterolemia has hepatic low-density lipoprotein receptors that are assumed to be half as many as in normal rabbits. CS-514 alone lowered plasma low-density lipoprotein cholesterol levels by 50%, and in combination with cholestyramine, it lowered levels by 80%. The membrane-binding assay showed these drugs caused 1.5- and 1.8-fold increases in the number of hepatic low-density lipoprotein receptors, respectively. We also measured the amount of mRNA for low-density lipoprotein receptor by S1 nuclease protection assay in the same livers as above. These drugs induced mutant mRNA for the low-density lipoprotein receptor, which has an in-flame deletion of 12 nucleotides, as well as normal receptor mRNA. CS-514 alone produced a 1.8-fold increase in the amount of mRNA for the normal receptor and a 2.3-fold increase for the mutant mRNA, whereas CS-514 in combination with cholestyramine produced 1.9- and 3.1-fold increases, respectively. We conclude that CS-514 induces mRNA for the low-density lipoprotein receptor, subsequently increasing the receptor protein in the liver, and then reduces the levels of plasma cholesterol, and that the induction is augmented when the drug is administered in combination with cholestyramine.

Probucol does not affect lipoprotein metabolism in macrophages of Watanabe heritable hyperlipidemic rabbits.

We recently reported that the antioxidant action of probucol inhibited the progression of atherosclerosis in Watanabe heritable hyperlipidemic (WHHL) rabbits. In this study, we investigated another possible action of probucol: its action as an antiatherogenic agent on macrophages. When WHHL rabbit peritoneal macrophages were pre-incubated in vitro with probucol and then incubated with several atherogenic lipoproteins, the incorporation of the lipoproteins was not significantly prevented. In the case of mouse peritoneal macrophages, pre-incubation with probucol showed slight, although not statistically significant, changes in the amount of lipoprotein incorporations. We also used macrophages obtained from mice and WHHL rabbits fed with probucol, but the amount of uptake of lipoproteins by these cells was not less than that by control macrophages. Furthermore, to investigate the incorporation of atherogenic lipoproteins into these cells, we prepared probucol-containing macrophages; however, probucol in macrophages failed to prevent the uptake of such lipoproteins. In conclusion, probucol did not prevent foam cell transformation of macrophages of WHHL rabbit or mice directly, and the effect of probucol against atherogenesis in WHHL rabbits was due mainly to its inhibitory effect on the oxidative modification of low density lipoprotein, as previously reported.

Different expression of modified low density lipoprotein receptors in rabbit peritoneal macrophages and Kupffer cells

We have previously reported that mouse peritoneal macrophages have three types of modified low density lipoprotein (LDL) receptors. One is specific for acetylated LDL (Ac-LDL), the second is for oxidized LDL (Ox-LDL), and the third recognizes both (Arai, H. et al. (1989) Biochem. Biophys. Res. Commun. 159, 1375-1382). In the current study, the characteristics of modified LDL receptors in rabbit peritoneal macrophages and Kupffer cells from rabbits were investigated. Cross-competition studies of the degradation assay between Ox-LDL and Ac-LDL in rabbit peritoneal macrophages showed that the degradation of 125I-labeled Ox-LDL was almost completely inhibited by an excess amount of unlabeled Ac-LDL. On the other hand, an excess amount of unlabeled Ox-LDL suppressed 125I-labeled Ac-LDL degradation only partially. In contrast, in Kupffer cells an excess amount of unlabeled Ox-LDL inhibited the degradation of 125I-labeled Ac-LDL almost completely, whereas the degradation of 125I-labeled Ox-LDL was inhibited only partially by Ac-LDL. Scatchard analysis of binding assay showed that rabbit peritoneal macrophages have a single class of receptor for Ox-LDL, which binds maximally 0.31 microgram/mg cellular protein (Bmax) with an apparent dissociation constant (Kd) of 19.3 micrograms/ml, and two classes of receptors for Ac-LDL; one with high affinity (Bmax 0.025 microgram/mg cellular protein, Kd 0.040 micrograms/ml) and the other with low affinity (Bmax 0.08 microgram/mg cellular protein, Kd 11.31 micrograms/ml). On the other hand, Kupffer cells have two classes for Ox-LDL; one is a high affinity receptor (Bmax 0.53 microgram/mg cellular protein, Kd 0.99 microgram/ml) and the other is a low affinity receptor (Bmax 3.71 micrograms/mg cellular protein, Kd 16.2 micrograms/ml) and a single class for Ac-LDL (Bmax 0.60 microgram/mg cellular protein, Kd 7.24 micrograms/ml). These results indicate that rabbit peritoneal macrophages have two kinds of modified LDL receptors; one is specific for Ac-LDL, and the other recognizes both Ox-LDL and Ac-LDL.

Induction of Endothelial Platelet-Derived Growth Factor-B-Chain and Intercellular Adhesion Molecule-1 by Lysophosphatidylcholinea

Lysophosphatidylcholine (lyso-PC) is a major phospholipid component of atherogenic lipoproteins. Lyso-PC has been shown to differentially upregulate the adhesion molecules, such as VCAM-1 and ICAM-1, as well as smooth muscle growth factors, such as PDGF-A, B chains and HB-EGF gene expression in various cultured endothelial cells. In this paper, we demonstrate increased expression of cell- and matrix-associated forms of PDGF-B protein elicited by lyso-PC and further characterized potential signal transduction mechanisms responsible for lyso-PC-induced human umbilical vein endothelial cell. Cycloheximide inhibited PDGF-B but not ICAM-1 mRNA induction by lyso-PC, suggesting the dependence on de novo protein synthesis for PDGF-B, but not ICAM-1. A protein kinase C (PKC) inhibitor did not block lyso-PC-induced increases in PDGF-B or ICAM-1 mRNA. The elevated level of cAMP blocked both PDGF-B and ICAM-1 upregulation by lyso-PC. However cAMP-elevating agents did not suppress ICAM-1 upregulation by PMA. Taken together, PDGF-B and ICAM-1 gene induction by lyso-PC may involve different signaling mechanisms; however, both appear to be independent of PMA-regulatable PKC activation but are suppressed by increased levels of intracellular cAMP.

Probucol pretreatment enhances the chemotaxis of mouse peritoneal macrophages.

To investigate the effects of probucol on macrophage chemotaxis, we preincubated mouse peritoneal macrophages with probucol for 20 hours in vitro and using a modified Boyden chamber system compared their chemotactic responses with those of control macrophages that were preincubated with vehicle. Probucol pretreatment enhanced the macrophage chemotactic responses to zymosan-activated serum, acetylated low density lipoprotein (LDL), and native LDL. Probucol pretreatment also enhanced the basal migration observed when there was no stimulant in the lower chamber of a modified Boyden chamber. The chemoattracting potency of native LDL was weaker than that of zymosan-activated serum in control macrophages; however, both substances became equally potent when the macrophages were preincubated with probucol. The degree of the enhancement to native LDL after probucol preincubation reached fourfold to eightfold. The fashion of the enhanced migration of macrophages to native LDL after preincubation with probucol was predominantly chemotactic rather than chemokinetic. Time-course experiments revealed that it took more than 12 hours of probucol preincubation to show clearly enhanced macrophage chemotaxis to native LDL. Macrophages preincubated with probucol together with cycloheximide showed markedly reduced chemotaxis compared with macrophages preincubated only with probucol. Probucol pretreatment also enhanced macrophage chemotactic responses to high density lipoprotein, oxidized LDL, and lipoprotein-deficient serum.(ABSTRACT TRUNCATED AT 250 WORDS)