Follistatin-like 1, a secreted muscle protein, promotes endothelial cell function and revascularization in ischemic tissue through a nitric-oxide synthase-dependent mechanism.

Myogenic Akt signaling coordinates blood vessel recruitment with normal tissue growth. Here, we investigated the role of Follistatin-like 1 (Fstl1) in the regulation of endothelial cell function and blood vessel growth in muscle. Transgenic Akt1 overexpression in skeletal muscle led to myofiber growth that was coupled to an increase in muscle capillary density. Myogenic Akt signaling or ischemic hind limb surgery led to the induction of Fstl1 in muscle and increased circulating levels of Fstl1. Intramuscular administration of an adenoviral vector expressing Fstl1 (Ad-Fstl1) accelerated flow recovery and increased capillary density in the ischemic hind limbs of wild-type mice, and this was associated with an increase in endothelial nitric oxide synthase (eNOS) phosphorylation at residue Ser-1179. In cultured endothelial cells, Ad-Fstl1 stimulated migration and differentiation into network structures and inhibited apoptosis under conditions of serum deprivation. These cell responses were associated with the activating phosphorylation of Akt and eNOS. Conversely, transduction with dominant-negative Akt or LY294002 blocked Fstl1-stimulated eNOS phosphorylation and inhibited Fstl1-stimulated cellular responses. Treatment with the eNOS inhibitor N(G)-nitro-L-arginine methyl ester also reduced endothelial cell migration and differentiation induced by Ad-Fstl1. The stimulatory effect of Ad-Fstl1 on ischemic limb reperfusion was abolished in mice lacking eNOS. These data indicate that Fstl1 is a secreted muscle protein or myokine that can function to promote endothelial cell function and stimulates revascularization in response to ischemic insult through its ability to activate Akt-eNOS signaling.

Contribution of Cdc42 to cholesterol efflux in fibroblasts from Tangier disease and Werner syndrome.

Atherosclerotic cardiovascular disease is a life-threatening disorder. Cholesterol efflux from the cells is the rate-limiting step in regulating the intracellular cholesterol content as well as raft structure in the plasma membrane. The defect of cholesterol efflux leads to the development of atherosclerosis. Tangier disease (TD), a hereditary high-density lipoprotein deficiency, is characterized by the presence of defective cellular cholesterol efflux. Using the cDNA subtraction technique, we found that expression of Cdc42 was decreased markedly in fibroblasts and macrophages from patients with TD. Madin-Darby canine kidney cells expressing the dominant-negative form of Cdc42 had a reduced lipid efflux; inversely, cells expressing the active form had increased efflux. Furthermore, we found that cellular lipid efflux was defective and Cdc42 was reduced in fibroblasts from a premature aging disorder, Werner syndrome. Complementation experiments using an adenovirus carrying wild-type Cdc42 successfully corrected impaired lipid efflux in Werner syndrome cells. We concluded that Cdc42 may play important roles in cellular cholesterol efflux and that dysregulation of this type of RhoGTPase might lead to the development of atherosclerotic cardiovascular disease.

Senescent phenotypes of skin fibroblasts from patients with Tangier disease.

Tangier disease (TD) is characterized by a deficiency of high density lipoprotein (HDL) in plasma and patients with TD have an increased risk for coronary artery disease (CAD). Recently, we reported that fibroblasts from TD exhibited large and flattened morphology, which is often observed in senescent cells. On the other hand, data have accumulated to show the relationship between cellular senescence and development of atherosclerotic CAD. The aim of the present study was to investigate whether TD fibroblasts exhibited cellular senescence. The proliferation of TD fibroblasts was gradually decreased at population doubling level (PDL) approximately 10 compared with control cells. TD cells practically ceased proliferation at PDL approximately 30. DNA synthesis was markedly decreased in TD fibroblasts. TD cells exhibited a higher positive rate for senescence-associated beta-galactosidase (SA-beta-gal), which is one of the biomarkers of cellular senescence in vitro. These data showed that TD cells reached cellular senescence at an earlier PDL compared with controls. Although, there was no difference in the telomere length of fibroblasts between TD and controls at the earlier passage (PDL 6), the telomere length of TD cells was shorter than that of controls at the late passage (PDL 25). Taken together, the current study demonstrates that the late-passaged TD fibroblasts showed senescent phenotype in vitro, which might be related to the increased cardiovascular manifestations in TD patients.

Increased lipid rafts and accelerated lipopolysaccharide-induced tumor necrosis factor-alpha secretion in Abca1-deficient macrophages.

Lipid rafts on the cell surface are believed to be very important as platforms for various cellular functions. The aim of this study was to know whether defective lipid efflux may influence lipid rafts on the cell surface and their related cellular functions. We investigated macrophages with defective lipid efflux from ATP binding cassette transporter A1-deficient (Abca1-KO) mice. Lipid rafts were evaluated by the following two novel probes: a biotinylated and protease (subtilisin Carlsberg)-nicked derivative of theta-toxin and a fluorescein ester of polyethylene glycol-derived cholesterol. Lipid rafts in Abca1-KO macrophages were increased, as demonstrated by both probes. Moreover, activities of nuclear factor kappaB, mRNA and intracellular distribution, and secretion of tumor necrosis factor-alpha (TNF-alpha) were examined after stimulation by lipopolysaccharides (LPSs). LPS-induced responses of the activation of nuclear factor kappaB and TNF-alpha were more prompt and accelerated in the Abca1-KO macrophages compared with wild-type macrophages. Modification of lipid rafts by cyclodextrin and nystatin corrected the abnormal response, suggesting an association between the increased lipid rafts and abnormal TNF-alpha secretion. We report here that Abca1-KO macrophages with defective lipid efflux exhibited increased lipid rafts on the cell surface and accelerated TNF-alpha secretion.

Probucol enhances the expression of human hepatic scavenger receptor class B type I, possibly through a species-specific mechanism.

OBJECTIVE: Scavenger receptor class B type I (SR-BI) is a major receptor for high-density lipoproteins (HDL) in the liver, which is the terminus of reverse cholesterol transport. Overexpression of SR-BI attenuated experimental atherosclerosis in murine models, concomitant with a reduction in plasma HDL-cholesterol levels. Probucol is known to be a potent hypolipidemic drug to regress xanthoma formation and carotid atherosclerosis in conjunction with a marked reduction in HDL-cholesterol levels. The aim of the present study was to know the effect of probucol on the expression of SR-BI and the underlying mechanism. METHODS AND RESULTS: We found that probucol increased the expression of SR-BI proteins in in vitro human liver cells and an in vivo rabbit model, but not in wild-type C57Bl6 mice. The decay curve of SR-BI protein was markedly retarded in probucol-treated HepG2 cells in the presence of cycloheximide, indicating that probucol may stabilize human SR-BI protein. To determine the underlying mechanism for the observed species-specific effect, we conducted the following host-swap experiments, in which SR-BI was transfected or expressed in heterologous cells or hosts. Probucol did not increase human SR-BI protein in the liver of transgenic mice carrying the entire human SR-BI genome. Although probucol could stabilize even murine SR-BI, when transfected into a human cell line, HepG2, human SR-BI was not stabilized in a mouse hepatoma cell line, Hepa 1-6, treated with probucol. CONCLUSIONS: Probucol enhances hepatic SR-BI protein expression, possibly through species-specific stabilization of the protein.

Defective cholesterol efflux in Werner syndrome fibroblasts and its phenotypic correction by Cdc42, a RhoGTPase.

Werner syndrome (WS) is characterized by the early onset of senescent phenotypes including premature atherosclerotic cardiovascular diseases, although the underlying molecular mechanism for atherosclerosis has not been fully understood yet. Cholesterol efflux from the cells is the initial step of reverse cholesterol transport, a major protective system against atherosclerosis. The aim of the present study was to determine whether this crucial step may be altered in WS. We examined intracellular lipid transport and cholesterol efflux and the expression levels of its related molecules in skin fibroblasts obtained from patients with WS. Cholesterol efflux was markedly reduced in the WS fibroblasts in association with increased cellular cholesterol. Fluorescent recovery after photobleaching (FRAP) technique revealed that intracellular lipid transport around Golgi apparatus was markedly reduced when using a C6-NBD-Ceramide as a tracer. Cdc42 protein and its GTP-bound form were markedly reduced in the WS fibroblasts. The complementation of wild-type Cdc42 corrected cholesterol efflux, intracellular lipid transport, and cellular cholesterol levels in the WS fibroblasts. These data indicated that the reduced expression of Cdc42 may be responsible for the abnormal lipid transport, which in turn might be related to the cardiovascular manifestations in WS.

Retarded intracellular lipid transport associated with reduced expression of Cdc42, a member of Rho-GTPases, in human aged skin fibroblasts: a possible function of Cdc42 in mediating intracellular lipid transport.

OBJECTIVE: Many cell types in atherosclerotic lesions are thought to have various biological abnormalities, such as impaired lipid homeostasis and slow cell proliferation, which may be related to senescence at cellular and individual levels. One of the common characteristics of senescent cells in vitro is the alteration of actin cytoskeletons, which have been reported to be involved in the intracellular transport of lipids. Recently, we raised the hypothesis that Cdc42, which is a member of the Rho-GTPase family and is known to play an important role in actin dynamics, might be important in cellular lipid transport. METHODS AND RESULTS: In the present study, we found that the protein expression levels and GTP-binding activities of Cdc42 were decreased in aged human skin fibroblasts. Moreover, we found the intracellular kinetics of Golgi-associated lipids to be retarded in these cells, which was demonstrated by the fluorescence recovery after photobleaching (FRAP) technique and the use of N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-6-aminohexanoyl-D-erythro-sphingosine as a tracer. To correlate the decreased expression of Cdc42 with the retarded FRAP, we complemented the amount of wild-type c-myc-tagged Cdc42Hs (myc-Cdc42Hs-WT) by adenovirus-mediated gene transfer. We further tested the effect of the dominant-active form (myc-Cdc42Hs-DA, V12Cdc42Hs) or dominant-negative form (myc-Cdc42Hs-DN, N17Cdc42Hs) of Cdc42Hs on FRAP. Introduction of myc-Cdc42Hs-WT or myc-Cdc42Hs-DA recovered the retarded FRAP in the aged fibroblasts. Conversely, control fibroblasts infected with myc-Cdc42Hs-DN exhibited significantly retarded FRAP. CONCLUSIONS: These data clearly indicate that the expression of Cdc42, a small G protein, is decreased in the aged cells in close association with the retarded intracellular lipid transport. The present study demonstrates a possible function of Cdc42 in the mediation of intracellular lipid transport.

Dominant expression of ATP-binding cassette transporter-1 on basolateral surface of Caco-2 cells stimulated by LXR/RXR ligands.

ATP-binding cassette transporter-1 (ABCA1) is a cause of Tangier disease, which is a familial deficiency of plasma high density lipoproteins (HDL). This molecule is known to be expressed in the multiple tissues and organs including small intestines, liver, and macrophages in the blood vessels. Recent in vivo studies suggested that ABCA1 plays some roles in the flux of cholesterol in the intestines. One of the major questions to understand the roles of ABCA1 in the intestines is the expression pattern in the intestinal epithelial cells. To address this issue, we have investigated the expression and regulation of ABCA1 in Caco-2 cells cultured on Transwell as a model, especially focusing on possible polarized expression of ABCA1. The expression of ABCA1 was up-regulated during the differentiation and under the stimulation of LXR/RXR by the addition of 9-cis-retinoic acid (9-cis-RA) and 22-R-hydroxycholesterol (22-OH). Apolipoprotein-AI-mediated cholesterol efflux was dominant toward the basolateral side of polarized cells when stimulated by 9-cis-RA and 22-OH. The cell surface biotinylation experiment followed by Western blot analyses demonstrated a markedly dominant expression of ABCA1 on the basolateral surface, which was clearly confirmed by the confocal laser scanning microscopy. In conclusion, the present study demonstrates that ABCA1 is dominantly expressed on the basolateral surface of Caco-2 cells tested, suggesting that this molecule may play a role in the basolateral movement of cholesterol at least when stimulated by LXR/RXR ligands.

Expression and functional analyses of novel mutations of ATP-binding cassette transporter-1 in Japanese patients with high-density lipoprotein deficiency.

ATP-binding cassette transporter-1 (ABCA1) gene is mutated in patients with familial high-density lipoprotein deficiency (FHD). In order to know the molecular basis for FHD, we characterized three different ABCA1 mutations associated with FHD (G1158A/A255T, C5946T/R1851X, and A5226G/N1611D) with respect to their expression in the passaged fibroblasts from the patients and in the cells transfected with the mutated cDNAs. Fibroblasts from the all patients showed markedly decreased cholesterol efflux to apolipoprotein (apo)-Al. In the fibroblasts homozygous for G1158A/A255T, the immunoreactive mass of ABCA1 could not be detected, even when stimulated by 9-cis-retinoic acid and 22-R-hydroxycholesterol. In the fibroblasts homozygous for C5946T/R1851X, ABCA1 mRNA was comparable. Because the mutant ABCA1 protein (R1851X) was predicted to lack the epitope for the antibody used, we transfected FLAG-tagged truncated mutant (R1851X/ABCA1-FLAG) cDNA into Cos-7 cells, showing that the mutant protein expression was markedly reduced. The expression of N1611D ABCA1 protein was comparable in both fibroblasts and overexpressing cells, although cholesterol efflux from the cells was markedly reduced. These data indicated that, in the three patients investigated, the abnormalities and dysfunction of ABCA1 occurred at the different levels, providing important information about the expression, regulation, and function of ABCA1.