Tissue Factor Expression In Macrophages Research Articles

Tofacitinib Affects M1-like and M2-like Polarization and Tissue Factor Expression in Macrophages of Healthy Donors and IBD Patients.

Tofacitinib, as inhibitor of Janus kinases (JAK), interrupts the transmission of numerous pro-inflammatory cytokines involved in the pathogenesis of inflammatory bowel diseases (IBD). Therefore, tofacitinib provides a potent option to treat ulcerative colitis (UC). Besides the anti-inflammatory potential, inhibition of widespread JAKs carries the risk of side effects. Macrophages are involved in the form of different subtypes in inflammation, wound healing, and even coagulation. This study aimed to explore the balanced use of tofacitinib in M1-like as well as M2-like macrophages of healthy donors and patients with IBD. Monocytes of healthy donors and patients with chronic courses of IBD were obtained from blood samples. Macrophage colony-stimulating factor (M-CSF)-derived macrophages were treated with tofacitinib (1 µM, 5 µM, 10 µM) and polarized with either lipopolysaccharide and interferon (IFN)-γ towards M1-like-phenotype or with interleukin (IL)-4 towards M2-like-phenotype. ELISA and flow cytometry were used to evaluate cytokine levels and surface molecules. Tofacitinib had a modulating effect on M1-like macrophages whereby the effect on pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, IL-12, IL-23) was less pronounced than the induction of anti-inflammatory IL-10. However, during M2-like polarization tofacitinib impaired the development of the corresponding phenotype becoming evident through decreased IL-10 levels and CD206 expression in treated macrophages. In both phenotypes, tofacitinib strongly downregulated the expression of immunostimulatory molecules (CD80, CD86, CD83, CD40). Furthermore, a dose-dependent correlation between treatment with tofacitinib and expressed tissue factor was noticed. Tofacitinib influences both polarizations (M1/M2) and the expression of tissue factor in a dose-dependent manner.

Role of JAK2–STAT3 in TLR2‐mediated tissue factor expression

Tissue factor (TF) is a core protein with an essential function in the coagulation cascade that maintains the homeostasis of the blood vessels. TF not only participates in neointima formation, but also causes the development of atherosclerosis. This study investigated the mechanism regulating TF expression in macrophages using Pam3 CSK4 , a TLR2 ligand. Pam3 CSK4 induced TF expression in two types of macrophages (Raw264.7 and BMDM), but not in TLR2 KO mice derived BMDM. Pam3 CSK4 induced TF expression was inhibited by pretreatment with pan-JAK inhibitor or JAK2 inhibitor AG490. JAK2 knock-down by siRNA inhibited Pam3 CSK4 induced TF expression. Pam3 CSK4 stimulated STAT3 phosphorylation (S727), while STAT3 knock-down by siRNA reduced Pam3 CSK4 induced TF expression. These results suggest that Pam3 CSK4 induced TF expression is regulated by the JAK2-STAT3 signaling pathway. Pam3 CSK4 , unlike increased TF expression, significantly decreased RGS2 expression, while RGS2 overexpression decreased Pam3 CSK4 induced TF expression. Inhibition of TF by RGS2 WT did not occur in mutants with flawed RGS domains. We also investigated the correlation between RGS2 and STAT3 phosphorylation. RGS2 knock-down elevated Pam3 CSK4 induced STAT3 phosphorylation, but RGS2 overexpression had the opposite effect on STAT3 phosphorylation. These results suggest that, while Pam3 CSK4 induced TF expression is regulated by JAK2-STAT3 signaling, RGS2 is a negative regulator targeted to STAT3.

01 Tristetraprolin Post-Transcriptionally Regulates Tissue Factor Expression in Primary Human and Murine Macrophages

Background Although monocyte-derived tissue factor (TF) plays critical roles in atherothrombosis, little is known about its post-transcriptional regulation. Tristetraprolin (TTP) binds the 3′UTR of target mRNAs and promotes their degradation, with its function being negatively regulated by p38 MAPK. Whether TTP post-transcriptionally regulates TF is unknown. Methods We used human monocytes and bone marrow derived macrophages from TTP+/+ and TTP-/- mice. Procoagulant activity was determined using a clot turbimetric assay. mRNA decay was determined following transcriptional arrest using actinomycin D. TTP knockdown was achieved using siRNA transfection. RNA and protein interaction was determined using ribonucleoprotein (RNP) immunoprecipitation and RNA biotin pulldown assays. Results p38 inhibition with SB203580 and SB202190 (1µMM) reduced procoagulant activity, TF mRNA and protein expression in human macrophages (p Conclusions These data provide evidence, for the first time, that p38 and TTP post-transcriptionally regulate TF expression in macrophages. A better understanding of the post-transcriptional regulation of TF expression will provide novel insights into the interface between inflammation and thrombosis in vascular biology, and holds therapeutic potential.

Mycobacterium tuberculosis Infection and Tissue Factor Expression in Macrophages

A number of earlier studies reported the occurrence of thrombotic complications, particularly disseminated intravascular coagulation and deep vein thrombosis, in tuberculosis (TB) patients. The aberrant expression of tissue factor (TF), the primary activator of coagulation cascade, is known to be responsible for thrombotic disorders in many diseases including bacterial infections. Further, expression of TF by cells of the monocyte/macrophage lineage is also shown to contribute to the development and progression of local and systemic inflammatory reactions. In the present study, we have investigated whether Mycobacterium tuberculosis (Mtb) infection induces TF expression in macrophages, and various host and pathogenic factors responsible for TF expression. We have tested the effect of live virulent Mtb H37Rv, gamma-irradiated Mtb H37Rv (γ-Mtb) and various components derived from Mtb H37Rv on TF expression in macrophages. The data presented in the manuscript show that both live virulent Mtb and γ-Mtb treatments markedly increased TF activity in macrophages, predominantly in the CD14+ macrophages. Detailed studies using γ-Mtb showed that the increased TF activity in macrophages following Mtb treatment is the result of TF transcriptional activation. The signaling pathways of TF induction by Mtb appears to be distinct from that of LPS-induced TF expression. Mtb-mediated TF expression is dependent on cooperation of CD14/TLR2/TLR4 and probably yet another unknown receptor/cofactor. Mtb cell wall core components, mycolyl arabinogalactan peptidoglycan (mAGP), phosphatidylinositol mannoside-6 (PIM6) and lipomannan (LM) were identified as factors responsible for induction of TF in the order of mAGP>PIM6>LM. A direct contact between bacteria and macrophage and not Mtb-released soluble factors is critical for TF induction by Mtb. In summary, our data show that Mtb induces TF expression in macrophages and Mtb signaling pathways that elicit TF induction require cooperation of multiple receptors, co-receptors/co-factors including Toll-like receptors. The importance of TF in granuloma formation and containment of Mtb is discussed.

Mycobacterium Tuberculosis infection and Tissue Factor Expression in Macrophages

Abstract 1198Tissue factor (TF), the primary initiator of the coagulation cascade, is normally absent from cells that regularly come in contact with blood such as endothelial cells and monocytes yet could be induced in these cells in a variety of pathological conditions. The aberrant expression of TF by cells of the monocyte/macrophage lineage is thought to be a major contributor to thrombotic disorders and inflammation. Macrophages play a central role in the innate immune response essential for host defense against pathogenic infections. Tuberculosis (TB), a fatal disease caused by Mycobacterium tuberculosis (Mtb), affects nearly one third of the world’s population. A number of studies have reported the presence of thrombotic complications, particularly disseminated intravascular coagulation (DIC) in TB patients. However, it is unclear how Mtb infection causes DIC or other thrombotic disorders as mycobacteria are not known to produce endotoxins or exotoxins that otherwise initiate the clotting cascade. In the present study, we have investigated whether Mtb infection induces TF expression in macrophages and various host and pathogenic factors responsible for TF expression. We have tested the effect of live virulent Mtb H37Rv, gamma-irradiated Mtb H37Rv (γ-Mtb) and various components derived from Mtb H37Rv on TF expression in macrophages. Exposure of human monocyte-derived macrophages (MDM) to live virulent Mtb H37Rv (at 1 to 5 bacteria/macrophage) or γ-Mtb H37Rv (10 μg/ml) markedly increased TF expression in MDM (5 to 20-fold increase compared to untreated MDM). TF expression in macrophages in response to Mtb is predominantly higher in CD14hi (CD14+) macrophages. Although CD14loCD16hi (CD16+) macrophages also expressed TF in response to Mtb, the level of TF induction in these cells was much lower. γ-Mtb induced TF expression in macrophages much more robustly than E.coli-derived LPS. The γ-Mtb-mediated induction of TF expression in macrophages peaked around 9 h and was sustained throughout 48 h, which markedly varies from the kinetics of LPS-induced TF expression, which peaks between 3–6 h and then reduced down to basal level by 18–24 h. To identify the potential macrophage receptor(s) responsible for the induction of TF by Mtb, MDM were pre-treated with antibodies against CD14 and Toll-like receptors (TLR) before they were exposed to γ-Mtb. Although CD14 antibodies markedly inhibited LPS-induced TF activity, they only suppressed Mtb-induced TF activity minimally. Pretreatment of MDMs with TLR2 or TLR4 antibodies had no significant effect on γ-Mtb-induced TF expression. However, combining the CD14 antibodies together with TLR2 and TLR4 antibodies showed nearly a 50% reduction in γ-Mtb-induced TF activity. These data indicate that a cooperative action of multiple receptors and signaling pathways may be responsible for the robust and sustained induction of TF expression. In order to identify the specific component(s) of γ-Mtb that are responsible for TF induction, we have treated MDMs with various subcellular fractions or purified components derived from Mtb H37Rv. The whole cell lysate of Mtb, cell wall, cell membrane and culture filterate proteins induced TF activity to varying degrees. Of all the purified components tested, the mycobacterial cell wall core component mycolyl arabinogalactan peptidoglycan (mAGP), phosphatidylinositol mannoside-6 (PIM6) and lipomannan (LM) elicited induction of TF protein and activity in the order of mAGP > PIM6 > LM. It is interesting to note that the treatment of MDMs with individual components, mycolic acid, arabinogalactan and peptidoglycan of mAGP complex did not induce TF expression, indicating that the structure of mAGP may be required for recognition by pattern recognition receptors on macrophages. In summary, our data show that Mtb induces TF expression in macrophages, probably through a novel mechanism. Although Mtb-induced TF expression in monocytes/macrohages may lead to systemic thrombotic disorders through the release of TF containing microparticles, it may be the localized and sustained expression of TF on macrophages that internalize the infectious bacteria at the site of infection in lung is responsible for granuloma formation in tuberculosis patients that prevents the spread of the bacteria. Disclosures:No relevant conflicts of interest to declare.

Xuezhikang, Extract of Red Yeast Rice, Inhibited Tissue Factor and Hypercoagulable State Through Suppressing Nicotinamide Adenine Dinucleotide Phosphate Oxidase and Extracellular Signal-regulated Kinase Activation

Xuezhikang, extract of red yeast rice, is a traditional Chinese medicine with multiple cardioprotective effect. It contains a family of naturally occurring statins, such as lovastatin. Tissue factor (TF) is overexpressed in macrophages of lipid core plaques, which display high procoagulant activity and seem to be a potentially target for anti-atherothrombosis. Therefore, the purpose of this study was to explore the effect and possible molecular mechanisms of xuezhikang on inhibiting TF expression and hypercoagulable state and the differences compared with lovastatin. Our results showed that xuezhikang significantly suppressed oxidized low-density lipoprotein-induced TF expression in macrophages in a concentration-dependent manner. Xuezhikang reduced nicotinamide adenine dinucleotide phosphate oxidase activity by decreasing membrane translocation of p47 through inhibition of extracellular signal-regulated kinase 1/2 activation. Nicotinamide adenine dinucleotide phosphate inhibitor (diphenyleneiodonium) also inhibited the oxidized low-density lipoprotein-induced TF expression, similar to the effects of xuezhikang. Furthermore, consistent with the severity of aortic atherosclerosis, xuezhikang (300 mg·kg·d) significantly reduced blood coagulation activation and TF expression in high-cholesterol diet-induced atherosclerotic rats. In addition, xuezhikang was more potent than lovastatin on inhibiting the expression of TF and nicotinamide adenine dinucleotide phosphate oxidase activation. These observations provide evidences that inhibition of xuezhikang on hypercoagulation and TF expression may partly account for its cardioprotective benefits.

Cardiac RAGE in Sepsis

See related article, pages 1239–1246 Sepsis is a systemic inflammatory response syndrome triggered by infection. The hallmarks of clinical sepsis are a broad range of systemic and organ function aberrations, including core temperature (hypothermia or hyperthermia), cardiac (rate and contractility), and respiratory and hematologic perturbations. When sepsis results in at least 1 organ failure or dysfunction, it is classified as severe sepsis. Severe sepsis with hypotension unresponsive to fluid resuscitation defines septic shock. Sepsis syndrome afflicts almost 750 000 patients in the United States each year, at a cost of almost $17 billion, and causes more than 200 000 deaths annually. The incidence of sepsis syndrome continues to rise along with the increase in life span and several other important risk factors. Sepsis without organ dysfunction is a relatively benign condition, and spontaneous recovery with conservative measures results in low in-hospital mortality (5% to 10%). Severe sepsis and septic shock carry high mortality, 30% to 50%, in spite of all that modern treatment offers.1–3 Genetic predisposing factors have been proposed including mutations in cytokine genes such as tumor necrosis factor (TNF)-α, interleukin (IL)-1, and IL-6. Most of these polymorphisms, implicated in increasing severity of sepsis, are associated with increases in these proinflammatory cytokines. Abnormalities in Toll-like receptor-4 and its signaling kinase, IRAK-4, have been associated with worse outcomes. The alarming increase in pathogens resistant to antibiotics that, in the past, were very effective in prevention and treatment of sepsis no doubt contributes to increases in severe sepsis prevalence in the elderly. The key mechanisms believed to initiate and propagate …

Protease Signaling in Severe Systemic Inflammation.

The tissue factor (TF) pathway drives disseminated intravascular coagulation in sepsis. Although extensive in vitro data demonstrate proinflammatory effects of protease activated receptor (PAR) signaling, it remains unclear whether PAR signaling plays a significant role in severe systemic inflammation. In a murine model of severe, 75–90% lethal endotoxemia, we addressed the interplay between intravascular coagulation activation (based on thrombin-antithrombin complexes) and inflammatory exacerbation (based on inflammatory cytokines IL6, IL1b and MCP1) downstream of PAR signaling. TF cytoplasmic domain deleted mice displayed increased TF expression in macrophages and enhanced systemic coagulation activation. However, these mice resolved inflammation earlier than wild-type controls. Thus, intravascular coagulation activation per se does not predict survival. Rather, genetic deletion of PAR2 demonstrated that PAR2 signaling increased survival in TF cytoplasmic domain-deleted mice. PAR2 deficiency alone initially attenuated inflammatory cytokine production, but did not prevent progressive exacerbation of coagulation and inflammation, further evidence for protective PAR2 signaling in advanced, severe endotoxemia. In contrast, PAR1-deficiency had no effect on initial inflammation and coagulation, but at late stages attenuated inflammation and prolonged survival. In addition, PAR1-deficiency produced a synergistic survival benefit with TF cytoplasmic domain deletion. These experiments show that coagulation protease signaling both regulates and amplifies severe systemic inflammation. Strategies to target TF cytoplasmic domain signaling may therefore be beneficial to reduce sepsis lethality in combination with therapeutic blockade of proinflammatory PAR1 signaling.

Liver X receptor agonists inhibit tissue factor expression in macrophages

Exposure of blood to tissue factor (TF) rapidly initiates the coagulation serine protease cascades. TF is expressed by macrophages and other types of cell within atherosclerotic lesions and plays an important role in thrombus formation after plaque rupture. Macrophage TF expression is induced by pro-inflammatory stimuli including lipopolysaccharide (LPS), interleukin-1beta and tumor necrosis factor-alpha. Here we demonstrate that activation of liver X receptors (LXRs) LXRalpha and LXRbeta suppresses TF expression. Treatment of mouse peritoneal macrophages with synthetic LXR agonist T0901317 or GW3965 reduced TF expression induced by pro-inflammatory stimuli. LXR agonists also suppressed TF expression and its activity in human monocytes. Human and mouse TF promoters contain binding sites for the transcription factors AP-1, NFkappaB, Egr-1 and Sp1, but no LXR-binding sites could be found. Cotransfection assays with LXR and TF promoter constructs in RAW 264.7 cells revealed that LXR agonists suppressed LPS-induced TF promoter activity. Analysis of TF promoter also showed that inhibition of TF promoter activity by LXR was at least in part through inhibition of the NFkappaB signaling pathway. In addition, in vivo, LXR agonists reduced TF expression within aortic lesions in an atherosclerosis mouse model as well as in kidney and lung in mice stimulated with LPS. These findings indicate that activation of LXR results in reduction of TF expression, which may influence atherothrombosis in patients with vascular disease.

HMG CoA reductase inhibitors and the risk of venous thrombosis among postmenopausal women

HMG CoA reductase inhibitors and the risk of venous thrombosis among postmenopausal women

Chlamydia pneumoniae induces tissue factor expression in mouse macrophages via activation of Egr-1 and the MEK-ERK1/2 pathway.

Recent studies have suggested that infection with Chlamydia pneumoniae (C pneumoniae) may contribute to the instability of atherosclerotic plaques and thrombosis and is associated with acute coronary events. Tissue factor (TF), a potent prothrombotic molecule, is expressed by macrophages and other cell types within atherosclerotic lesions and plays an essential role in thrombus formation after plaque rupture. Therefore the effects of C pneumoniae on induction of TF expression in macrophages were investigated. Infection of RAW mouse macrophages with C pneumoniae induced a time-dependent increase in procoagulant activity, expression of TF protein, and TF mRNA. C pneumoniae infection stimulated increased binding of nuclear proteins to the consensus DNA sequence for Egr-1, a key response element within the TF promoter, and increased the expression of Egr-1 protein. Transient transfections of RAW cells with mutated TF promoter constructs showed that the Egr-1 binding region is an important transcriptional regulator of C pneumoniae-induced TF expression. Furthermore, C pneumoniae-stimulated phosphorylation of ERK1/2 and Elk-1 and pharmacological inhibition of mitogen-activated protein kinase activity reduced the expression of TF and Egr-1. Antibody and polymyxin B blocking of the Toll-like receptor 4 (TLR4) partially reduced the C pneumoniae-induced expression of TF and Egr-1. In conclusion, the C pneumoniae-induced increase in TF expression in macrophages is mediated in part by Egr-1, signaling through TLR4, and activation of the MEK-ERK1/2 pathway.

Expression of tissue factor, the clotting initiator, on macrophages in Plasmodium falciparum-infected placentas.

The expression of tissue factor (TF), the initiator of the clotting system, was investigated by immunohistochemical staining for its role in clotting mechanisms of Plasmodium falciparum-infected placenta. Most mononuclear cells in the intervillous space of infected placentas stained with an anti-TF monoclonal antibody (MAb) and were positive for antimacrophage MAb. The intervillous space of infected placentas had significant fibrin deposition. In contrast, only small amounts of leukocytes, TF-positive cells, and fibrin were seen in the intervillous space in noninfected placentas. These results indicate that macrophages accumulated in infected placentas express TF and that subsequent perivillous fibrin clot formation leads to a narrowing and plugging of the intervillous space and disturbance of the blood supply. Macrophage TF expression in placentas could be associated with retarded placental growth and low birth weight in malaria infection and should be further investigated.

15-Deoxy-Δ12,14-Prostaglandin J2 Inhibits Tissue Factor Expression in Human Macrophages and Endothelial Cells: Evidence for ERK1/2 Signaling Pathway Blockade

Basic and clinical evidence has provided insight into the molecular events that link inflammation and coagulation. Increased expression of tissue factor (TF) by circulating and vascular cells has been indicated as responsible for the thrombotic complications associated with acute and chronic inflammation. TF is indeed inducible in circulating and vascular cells by cytokines and bacterial lipopolysaccharide (LPS) and its expression triggers the coagulation. The cyclopentenone prostaglandins are naturally occurring prostaglandin D2 (PGD2) derivatives that comprises prostaglandin J2 (PGJ2) and its metabolites delta12-PGJ2 and 15-deoxy- delta12,14-prostaglandin J2 (15d-PGJ2). These compounds, detected in vivo in a later phase of the inflammatory response, are characterized by anti-inflammatory activity and participate to the resolution of inflammation. We have here investigated the effect of 15d-PGJ2 on TF expression in human macrophages and endothelial cells (HUVEC). Our results indicate that 15d-PGJ2 down-regulates LPS- and TNFalpha-induced TF activity, protein and mRNA through inhibition of TF gene transcription. The effect of 15d-PGJ2 is targeted to the NF-kappaB/I-kappaB pathway and to the mitogen activated protein kinase ERK1/2. A role of PPAR-gamma activation in TF inhibition by 15d-PGJ2 was excluded. We conclude that 15d-PGJ2 negatively affects TF expression in macrophages and endothelial cells through a PPARgamma-independent mechanism. This down-regulation may be crucial to limit excessive blood clotting activation in immuno-inflammatory diseases.

The role of tyrosine phosphorylation in lipopolysaccharide- and zymosan-induced procoagulant activity and tissue factor expression in macrophages.

The expression of surface procoagulants by exudative macrophages represents an important mechanism underlying local fibrin deposition at sites of extravascular inflammation. The present studies investigated the contribution of tyrosine phosphorylation to the generation of macrophage procoagulant activity (PCA) and tissue factor expression in response to proinflammatory stimuli. Both lipopolysaccharide (LPS) and zymosan rapidly stimulated tyrosine phosphorylation in elicited murine peritoneal macrophages. This effect was prevented by the tyrosine kinase inhibitors genistein and herbimycin and augmented by the addition of the phosphotyrosine phosphatase inhibitor vanadate. The vanadate-mediated rise in phosphotyrosine accumulation was abrogated by the use of diphenylene iodonium, an inhibitor of the respiratory burst oxidase, suggesting a role for peroxides of vanadate as contributors to the tyrosine phosphorylation. This notion was supported by the finding that vanadyl hydroperoxide markedly increased the accumulation of phosphotyrosine residues. To define the role of tyrosine phosphorylation in the induction of macrophage PCA by LPS, the effects of tyrosine kinase inhibition by genistein and herbimycin were investigated. Both agents inhibited the expression of macrophage PCA. Further, Northern blot analysis with the cDNA probe for murine tissue factor indicated that the inhibition occurred at the mRNA level or earlier. Since vanadate augmented phosphotyrosine accumulation, it was hypothesized that it might enhance generation of macrophage products. However, vanadate reduced induction of PCA in response to LPS. By contrast, vanadate augmented basal prostaglandin E2 (PGE2) release and stimulated PGE2 release by macrophages. Indomethacin prevented the increase in PGE2 but only partially restored normal levels of PCA. The effect of vanadate on tissue factor expression appeared to be posttranscriptional. These studies thus demonstrate, by functional Western blotting and Northern blotting techniques, that tyrosine phosphorylation plays a role in the regulation of macrophage PCA and tissue factor expression in response to proinflammatory stimuli.

Vastatins inhibit tissue factor in cultured human macrophages. A novel mechanism of protection against atherothrombosis.

We examined the effect of fluvastatin, the first entirely synthetic 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor that is structurally different from other vastatins, on tissue factor (TF) expression in human macrophages spontaneously differentiated in culture from blood monocytes. Fluvastatin decreased TF activity in a dose-dependent manner (1 to 5 mumol/L) in both unstimulated and lipopolysaccharide-stimulated macrophages, and this reduction paralleled the decrease in immunologically recognized TF protein. The same results were obtained with another lipophilic vastatin, simvastatin, but not with hydrophilic pravastatin. The reduction in TF expression was also observed in macrophages enriched in cholesterol after exposure to 50 micrograms/mL acetylated low density lipoprotein. The inhibitory effect of fluvastatin on TF activity and antigen was fully reversible by coincubation with 100 mumol/L mevalonate or 10 mumol/L all-trans-geranylgeraniol but not with dolichol, farnesol, or geraniol. Suppression of TF antigen and activity was accompanied by a diminution in TF mRNA levels, which was completely prevented by mevalonate. Furthermore, fluvastatin impaired bacterial lipopolysaccharide-induced binding of c-Rel/p65 heterodimers to a kappa B site in the TF promoter, indicating that this drug influences induction of the TF gene. We conclude that lipophilic vastatins inhibit TF expression in macrophages, and because this effect is prevented by mevalonate and geranylgeraniol, a geranylgeranylated protein plays a crucial role in the regulation of TF biosynthesis. The suppression of TF in macrophages by vastatins indicates a potential mechanism by which these drugs interfere with the formation and progression of atherosclerotic plaque as well as thrombotic events in hyperlipidemic patients.