Impact of different low-dose ritonavir regimens on lipids, CD36, and adipophilin expression.

We investigated the relationship between ritonavir concentrations and changes in lipids, vascular inflammation markers, CD36, and adipophilin expression in volunteers randomly assigned to groups receiving 100 mg of ritonavir once daily or twice daily. In both groups decreases in high-density lipoprotein (HDL) (6%, P = 0.010; and 10%, P < 0.001, respectively) and CD36 (14%, P = 0.012; and 16%, P = 0.006, respectively) and increases in the vascular inflammation marker sCD40L (12%, P = 0.008; 19%, P = 0.003, respectively) were seen. Increases in adipophilin (30%, P = 0.044) and triglycerides (32%, P = 0.044) were seen only in the group receiving ritonavir twice daily. The ritonavir concentration in the plasma correlated with changes in triglycerides, HDL, and adipophilin (r = 0.34, P = 0.030; r = 0.33, P = 0.040; and r = 0.4, P = 0.01, respectively).

Metabolic-inflammatory changes, and accelerated atherosclerosis in HIV patients: rationale for preventative measures.

Human immunodeficiency virus (HIV)-infected patients are at a significantly higher risk from coronary heart diseases (CHD) and myocardial infarction (MI) compared to gender- and age-matched non-infected individuals. Combination antiretroviral therapy (cART) has transformed a fatal illness into a chronic stable condition. However, cART induces metabolic abnormalities in HIV-infected patients, while its role in vascular atherosclerosis is still under investigation. The use of cART is linked to inflammation - a key mechanism in atherosclerotic progression and destabilisation that precedes clinical events like MI. There is evidence of visceral fat abnormal distribution in HIV infected patients, and inflammatory changes in HIV infected patients drive the initiation, progression and, ultimately, thrombotic clinical complications induced by atherosclerosis. Visceral adipose tissue, a virtual factory for manufacturing pro-inflammatory mediators, affects the liver function. The inflamed liver promotes the development of pro-atherogenic dyslipidaemia. Pro-inflammatory cytokines released by adipocytes travel to the skeletal muscles and other peripheral tissues, worsening insulin sensitivity and leading to hyperglycaemia. Increased high sensitivity C-reactive protein (hs-CRP) inflammatory marker is associated with endothelial dysfunction in HIV-infected patients. Increased levels of monocytic nuclear factor kappa-B (NFkappa-B), a master switch in the inflammatory cascade, are documented in patients with elevated hs-CRP levels. It can be assumed that, as a result of NFkappa-B activation, hs-CRP up-regulates cytokines that contribute to MI by recruiting leukocytes and promoting thrombosis. This review focuses on the association of HIV-infection, metabolic abnormalities and known mechanisms involved in inducing accelerated atherosclerosis and inflammation in HIV-infected patients, as well as the role of lipid lowering agents in potentially preventing CHD.

The dialogue between endothelial cells and monocytes/macrophages in vascular syndromes.

The aim of this chapter is to present and identify potential pharmacological targets in endothelial cell-monocyte interactions leading to vascular syndrome and involving inflammation, coagulation, vascular remodelling and thrombosis. Increasing evidence is indicating that endothelial cells play a key role in atherothombosis by their capacity to attract, bind and allow the extravasation of monocytes to sites of inflammation. Surface expression and/or activation of constituent cell adhesion molecules (for e.g. P-selectin, E-selectin, ICAM-1, and VCAM-1) on endothelial cells together with chemokines such as CXCL8 (IL-8), Platelet-activating factor (PAF), CCL2 and CCL5 (Table 1) allow the rolling, adhesion and extravasation of monocytes. This review focuses on pharmacological targets implicated in endothelial cells interactions with monocytes/macrophages in vascular disease states and on cutting edge genomic tools for the identification and characterization of such targets.

Identification and characterization of two genes (MIP-1beta, VE-CADHERIN) implicated in acute rejection in human heart transplantation: use of murine models in tandem with cDNA arrays.

BACKGROUND: Genes and mechanisms of action involved in human acute rejection after allogeneic heart transplantation remain to be elucidated. The use of a murine allograft model in tandem with cDNA arrays and quantitative real-time polymerase chain reaction (Q-PCR) can greatly help in identifying key genes implicated in human heart acute rejection. METHODS AND RESULTS: Hearts from Balb/c mice were either not transplanted or transplanted heterotopically in the abdomen of Balb/c (isografts) and C57BL/6 (allografts) mice. Histological analysis showed acute rejection only in allografts. Total RNA was extracted from isografts (n=3), allografts (n=4), and not transplanted hearts (n=4); reverse transcribed; and labeled with P32. Each probe was hybridized to cDNA macroarrays. Eight genes were overexpressed and 7 genes were underexpressed in allografts compared with isografts. Macrophage inflammatory protein-1beta (MIP-1beta), an overexpressed gene, and VE-cadherin, an underexpressed gene, were validated by immunohistochemistry and Q-PCR in the murine models. Genes of interest, validated in the 3 murine groups, were then investigated in human heart tissues. Immunohistochemistry and Q-PCR performed on endomyocardial biopsies after heart transplantation showing no rejection (n=10) or grade IB (n=10) or IIIA (n=10) rejection, according to International Society of Heart and Lung Transplantation criteria, confirmed the results obtained from the murine model. CONCLUSIONS: We have demonstrated that the upregulation of MIP-1beta and downregulation of VE-cadherin may strongly participate in human acute heart rejection.

Acute hyperglycaemia induces changes in the transcription levels of 4 major genes in human endothelial cells: macroarrays-based expression analysis.

Hyperglycaemia, in insulin-dependent or independent diabetes mellitus, promotes endothelial cell (EC) dysfunction and is a major factor in the development of macro- or microvascular diseases. The mechanisms and the disease-related genes in vascular diseases resulting from hyperglycaemia are poorly understood. Macroarrays. bearing a total of 588 cDNA known genes, were used to analyze HUVEC gene transcription subjected to 25 or 5-mM glucose for 24 h. Isolated mRNA derived from treated first passage HUVEC were reverse transcribed, 32P labeled, and hybridized to the cDNA macroarrays. Results show that acute hyperglycaemia induces an up-regulation of seven major genes, four of which were not previously reported in the literature. Northern blot analyses, performed on these 4 genes, confirm macroarrays results for alphav, beta4, c-myc, and MUC18. Moreover, time course analysis (0, 2, 4, 8, 2, 16, 24 h) of alphav, beta4 c-myc, and MUC18 mRNA expression, observed by northern blot assays, showed a peak at time points situated between 2 to 8 h. The 3 other genes (ICAM-1, beta1, and IL-8), were shown by others to be significantly upregulated after glucose stimulation. Furthermore, ELISA assays performed on the supernatant of HUVEC culture medium showed a significant increase of IL-8 for cells treated with 25-mM compared to 5-mM glucose. Identified genes, upregulated in endothelial cells as a result of acute hyperglycaemia, may serve as therapeutic or diagnostic targets in vascular lesions present in diabetic patients. These results also demonstrate the use of cDNA macroarrays as an effective approach in identifying genes implicated in a diseased cell.

Glucose and insulin modulate the capacity of endothelial cells (HUVEC) to express P-selectin and bind a monocytic cell line (U937).

Diabetes mellitus is associated with increased prevalence of endothelial cell dysfunction and vascular diseases. Mechanisms leading to alterations in endothelial cell function are poorly understood. We report here that hyperglycaemia results in the expression of endothelial adhesion molecules involved in leukocyte adhesion and extravasation. Incubation of human umbilical cord endothelial cells (HUVEC) with 25 mM glucose induced the expression of P-selectin. This effect was reversed by the addition of 1 nM insulin. Moreover, increased ICAM-1 expression was observed upon HUVEC incubation with 25 mM glucose. Increased adhesion of U937 cells (a monocytic cell line) to endothelial cells cultured with 25 mM glucose was observed. High glucose-induced monocytes cell adhesion was inhibited by an anti-P-selectin monoclonal antibody (LYP20). These results show that high glucose concentration activates endothelial cells leading to monocytes adhesion providing further evidence that hyperglycaemia might be implicated in vessel wall lesions contributing to diabetic vascular disease.

Cell biotinylation provides a sensitive and effective detection technique for cellular adhesion assays: comparison with existing methods.

A simple, sensitive, colorimetric labelling method was devised to quantify cell adhesion, based on labelling the cell plasma membrane with biotin. This method was applied in adhesion assays, which involved the adherence of biotin-labelled, PMA-stimulated, U937 cells. These cells resemble monocytes, and were bound onto fibronectin-coated wells and to an ECV304 cell monolayer. The adherent U937 cells were detected by the addition of a peroxidase-conjugated anti-biotin antibody and a soluble colorimetric substrate. This assay is convenient, fast and sensitive, and able to detect 320-1000 U937 cells under the conditions described. This study has used titration assays to compare the biotinylation method with the existing cell quantification approaches of 51Cr radiolabelling and antibody dependent ELISA. Chromium labelling was the most sensitive technique, but we found the biotinylation method to be more convenient than radioactive labelling and more sensitive than conventional ELISA. Biotinylated cells were also used very effectively in a Stamper-Woodruff adhesion assay with U937 cells binding to histological sections of atherosclerotic plaques. The selective detection of the bound cells permitted automated quantitation by image analysis. Whole cell biotinylation may have wider applications in biological research.

ICAM-1 deficiency reduces atherosclerotic lesions in double-knockout mice (ApoE(-/-)/ICAM-1(-/-)) fed a fat or a chow diet.

Intercellular adhesion molecule (ICAM)-1, a major adhesion molecule, plays a critical role in the homing of leukocytes to sites of atherosclerotic lesions. However, very little is known on the role of ICAM-1 in initiating and perpetuating vascular lesions in ApoE(-/-) mice fed a chow or a fat diet. This study has investigated the mean aortic lesions in mice (C57BL6 background) with a single-knockout (ApoE(-/-)) or double-knockout (DKO; ApoE(-/-), ICAM-1(-/-)) fed a chow or a fat diet over a period of 3, 6, 15, and 20 weeks. A 3-fold reduction in lesion size was observed at all time points in DKO mice fed a chow diet. However, in DKO mice fed a fat diet, a marked reduction in the aortic lesion was observed at 3 and 15 weeks, which did not reach a significant level at 6 and 20 weeks. This study shows in essence that DKO mice are protected from developing significant lesions for up to 6 weeks when fed a chow diet and from 3 to 6 weeks when fed a fat diet. After 6 weeks, the lesion size of the DKO mice follows that of the single-knockout mice when fed a chow diet and gets to the same level in mice fed a fat diet. Plasma cholesterol levels were not altered as a result of ICAM-1 deficiency. These studies show that ICAM-1 is implicated in the formation and progression of atherosclerotic lesions.

Identification and cloning of a new gene (2A3-2), homologous to human translational elongation factor, upregulated in a proliferating rat smooth muscle cell line and in carotid hyperplasia.

Smooth muscle cells (SMCs), before migration and proliferation in the intima of the vessel wall, change from a normal contractile to a pathological proliferating phenotype. The molecular regulatory mechanisms implicated in such phenotypic changes remain poorly understood. In this study, using differential display, we have isolated for the first time a new gene (2A3-2) that is overexpressed in a rapidly proliferating, but not synthetic, rat SMC line. This was further confirmed by northern blot performed on the 2 cell types. Moreover, balloon catheter injury of rat carotids showed, by a virtual northern technique, an upregulation of this new gene in hyperplasia vessels. This new gene (2A3-2, 1.2 kb) was present in skeletal muscle, heart, aorta, lung, liver, kidney, and spleen. In addition, 5' rapid amplification of cDNA ends (5' RACE) allowed the cloning and sequencing of this 1.2-kb gene. Comparison of this newly identified gene sequence with data banks showed a strong homology to human and bovine mitochondrial translational elongation factor. The 2A3-2 gene, identified in this study, may play a vital role in the cascade of events implicated in switching SMC phenotype from a quiescent to a proliferate one.

Effect of a micronized purified flavonoid fraction on in vivo platelet functions in the rat.

The aim of this study was to investigate the effects of a micronized purified flavonoid fraction (MPFF) on in vivo rat platelet functions. Platelet aggregation and disaggregation were evaluated by a noninvasive, automated isotope monitoring system (AimsPlus). Indium-labeled platelets were injected into anesthetized rats and stimulated by adenosine diphosphate (ADP) (10 microg/kg, i.v.) or collagen (50 microg/kg, i.v.). Fibrinogen binding to ex vivo ADP-activated platelets was determined by flow cytometry. MPFF (100 mg/kg, p.o.) significantly reduced ADP-induced platelet aggregation (p<0.05) and increased platelet disaggregation (p<0.05) compared with controls. Moreover, MPFF inhibited collagen-induced platelet aggregation (p<0.001) and increased platelet disaggregation (p<0.01). In addition, fibrinogen binding to 2.5 or 5 microM ADP-stimulated platelets also was reduced significantly (p<0.05 and 0.01, respectively). These results show that MPFF inhibits in vivo rat platelet functions.

Effect of naftazone on in vivo platelet function in the rat.

The aim of this study was to investigate the in vivo effects of 50 mg/kg (i.p.) naftazone or ticlopidine on platelet functions in the rat. An automated isotope monitoring system (Aims plus) was used to determine the height of platelet aggregation and disaggregation (measured by the area under the curve, AUC) of 111indium-labelled platelets activated by ADP (10 microg/kg i.v.) or collagen (50 microg/kg i.v.). Fibrinogen-binding experiments were carried out with activated platelets in whole blood and measured by flow cytometry. Naftazone reduced the height of platelet aggregation induced by ADP compared with controls (P = 0.024). Ticlopidine-treated rats gave similar results (P = 0.008). Platelet disaggregation, following the aggregation induced by collagen, was significantly increased in naftazone-treated rats compared with controls (P = 0.003). Similar results were observed with ticlopidine-treated rats (P = 0.002). Fibrinogen binding to 2.5 or 5 microM ADP-stimulated platelets, from naftazone-treated rats, were significantly reduced compared with controls (P = 0.05 and 0.04 respectively). These results show that naftazone has similar inhibitory effects on rat platelet functions as ticloplidine. In conclusion, naftazone could be a useful agent to modulate platelet function in patients with cardiovascular disease.

Senescent human neutrophil binding to thrombospondin (TSP): evidence for a TSP-independent pathway of phagocytosis by macrophages.

This study investigated the interaction of apoptotic polymorphonuclear neutrophils (PMN) with thrombospondin (TSP), an important event mediating the clearance of apoptotic neutrophils by macrophages. We developed an in vitro assay to examine this interaction. Based on this assay, we found that apoptotic but not fresh PMN bound specifically to surface-immobilized TSP (33 +/- 0.03 x 10(3) cells/well) compared to fibrinogen, fibronectin or laminin (8.0 +/- 0.3 x 10(3) cells/well). Moreover, the binding was specific for surface bound but not soluble TSP and appeared to be divalent cation dependent, was not significantly inhibited by heparin and was sensitive to cycloheximide (CHX) treatment of senescent PMN (>90%) inhibition at 10 microM CHX). In contrast to the binding studies, phagocytosis of senescent PMN by macrophages was not affected by EDTA or cycloheximide. Phosphatidyl-L-serine liposomes, phospho-L-serine, glucosamine, galactosamine, and the acetylated sugars had no effect on phagocytosis. We conclude that: (i) there was specific binding of senescent human PMN to immobilized TSP, which is divalent cation dependent and requires new protein synthesis in the PMN during senescence; (ii) in addition to the recently defined TSP-dependent pathway, there is a TSP-independent pathway mediating phagocytosis of senescent PMN by macrophages. The identity of this pathway remains to be defined.

Mapping the epitope of a functional P-selectin monoclonal antibody (LYP20) to a short complement-like repeat (SCR 4) domain: use of human-mouse chimaera and homologue-replacement mutagenesis.

P-selectin (CD62P), an adhesion molecule localized in platelet alpha-granules and endothelial cell Weibel-Palade bodies, is rapidly expressed on the surface of activated cells. This adhesion molecule, a member of the selectin family, mediates leucocyte interactions with activated platelets or endothelial cells. The aim of this study was to identify and characterize the epitope of a functional blocking P-selectin monoclonal antibody (mAb), LYP20. LYP20 recognizes human or rat, but not mouse, P-selectin. Human/mouse chimaeras and wild-type constructs, modified by homologue replacement mutagenesis, were expressed in COS cells. Blocking anti-(P-selectin) mAbs (G1, G3 or CLB-thromb/6) were observed, by flow cytometry, to bind to the lectin-like domain. In contrast, LYP20 was found to bind to one of the P-selectin short complement-like repeats (SCR domain 4). Homologue replacement mutagenesis of SCR domain 4 (region delineated by amino acid residues 359-457) identified three amino acids (Cys412-->Ser, Cys416-->Ser or Arg415-->Lys) as being implicated in the LYP20 epitope. Deleting the region bearing the LYP20 epitope, from a wild-type CD62P construct, showed a decrease in polymorphonuclear leucocyte (PMNL) binding to transfected COS cells. In addition, mutation of one of the three amino acids, implicated in the LYP20 epitope, markedly affected PMNL binding to transfected COS cells but did not affect the binding of mAbs G1 and CLB-thromb/6. These results are the first to indicate (1) that a functional blocking anti-P-selectin mAb binds to SCR 4, a site other than the lectin-like/epidermal growth factor-like domain, and (2) that SCR domain 4 has a functional role in P-selectin-leucocyte interactions.

Thrombospondin induces dimerization of membrane-bound, but not soluble CD36.

CD36 is a cell surface receptor that has been shown to interact with a large variety of ligands including thrombospondin, collagen, Plasmodium falciparum-infected erythrocytes, apoptotic neutrophils, modified low density lipoproteins, anionic phospholipids and long chain fatty acids. A number of these CD36 ligands elicit the transduction of intracellular signals involved in cell activation and internalization of bound ligands. The engagement of CD36 possibly activates three cytosolic protein tyrosine kinases that are presumably associated with the C-terminal cytoplasmic tail of CD36. However, the mechanisms by which CD36 functions in ligand binding and signal transduction are poorly understood. In the present study, a membrane-bound and a truncated soluble form of CD36 were expressed in HeLa cells and analyzed by velocity-gradient centrifugation and chemical cross-linking. We show that membrane CD36 exists predominantly as a monomer but a homodimeric form is also found. In contrast, soluble CD36 sedimented in sucrose gradient as a monomer. However, when incubated with thrombospondin, the membrane form of CD36 predominantly sedimented as a dimer whereas soluble CD36 was monomeric. This study shows that thrombospondin has the ability to induce dimerization of CD36 and may be implicated in the signal transduction capacity of this adhesion molecule.

Vascular biology of CD36: roles of this new adhesion molecule family in different disease states.

The human CD36 antigen is a scavenger receptor and a celladhesion molecule expressed by platelets, monocytes and microvascular endothelial cells, among other cell types. It belongs to a new and growing family of integral membrane glycoproteins that recognize a wide range of ligands. CD36 has been implicated in hemostasis, thrombosis, malaria, inflammation, lipid metabolism and atherogenesis. Recently, significant advances in CD36 biology have been reported and new CD36-like proteins have been identified.

Characterization of two vaccinia CD36 recombinant-virus-generated monoclonal antibodies (10/5, 13/10): effects on malarial cytoadherence and platelet functions.

Extensive evidence is now available to show that the human CD36 antigen is a cellular receptor for thrombospondin, collagen, modified low-density lipoproteins, and long-chain fatty acids. Moreover, CD36 functions as one of the receptors that mediates the adhesion of Plasmodium-falciparum-infected erythrocytes to microvascular endothelium. In an attempt to identify new functional sites of this surface glycoprotein, anti-CD36 monoclonal antibodies were prepared using a vaccinia CD36 recombinant virus as a highly efficient immunization vector. In functional studies, one of these antibodies (clone 10/5) strongly inhibited the adhesion of P. falciparum-infected erythrocytes to purified CD36. This antibody also potentiated ADP-induced platelet activation. In contrast, a second antibody (clone 13/10) did not affect the cytoadherence of infected erythrocytes or platelet functions. Previous structural work performed on these antibodies has shown that clone 10/5 is directed against an epitope within the CD36 domain 155-183, whereas clone 13/10 interacts with another antigenic determinant defined by amino acids 30-76 [Daviet, L., Buckland, R., Puente Navazo, M. D. & McGregor, J. L. (1995) Biochem. J. 305, 221-224]. Taken together, these current studies show that: (a) the methodology of immunization using recombinant vaccinia virus is a powerful tool in the generation of monoclonal antibodies directed against polyimmunogenic membrane glycoproteins such as CD36; (b) the CD36 domain, recognized by clone 10/5 but not by 13/10, is functionnally important regarding the adhesion of P. falciparum-infected erythrocyte and CD36-dependent platelet activation.

Platelet/polymorphonuclear leukocyte interaction in dynamic conditions: evidence of adhesion cascade and cross talk between P-selectin and the beta 2 integrin CD11b/CD18.

Adhesion between platelets and polymorphonuclear leukocytes (PMN) is a key event in thrombosis and inflammation. Double color fluorescence-activated cell sorter (FACS) analysis was used to determine the extent and kinetics of adhesion of thrombin-activated platelets to resting or activated PMN when mixed cell populations were incubated in dynamic conditions. Activated platelets bound very rapidly to PMN. Mixed cell conjugates reached a maximum at 1 minute and were reversible within 10 minutes. Platelet/PMN adhesion required both Ca2+ and Mg2+ and was markedly increased by the presence of Mn2+. The latter made mixed cell conjugates stable up to 10 minutes. Adhesion of platelets required metabolic activity of PMN and was abolished by tyrosine kinase inhibitors. Furthermore, adhesion of platelets to PMN resulted in binding of a monoclonal antibody (MoAb 24) known as beta 2 integrins "activation reporter." When PMN were activated by exogenous stimuli, the adhesion of platelets was markedly increased: fMLP induced a rapid and transient effect, while PMA resulted in a slower, but stable, increase in mixed conjugates formation. The hypothesis that activated PMN beta 2 integrins are able to bind a counter-receptor on platelets was directly demonstrated by the increase of mixed cell conjugates following PMN treatment with KIM127 and KIM185, two anti-CD18 antibodies able to induce the active conformation of beta 2 integrins. Consistently, two other anti-CD18, as well as an anti-CD11b inhibitory antibody abolished platelet/PMN adhesion. PMN beta 2 integrin activation was not the only mechanism for activated platelet/PMN adhesion to occur: indeed, this phenomenon could also be inhibited by two anti-P-selectin antibodies. Resting platelets did not adhere to resting PMN, but markedly adhered to fMLP- or PMA-activated PMN. Resting platelet/fMLP-activated PMN adhesion was abolished by anti-CD18 antibodies, but not by anti-P-selectin antibodies. In conclusion, activated platelet/PMN interaction can be modeled as an adhesion cascade involving a P-selectin-dependent recognition step and a functional signal. The latter proceeds through tyrosine kinase activation and enables a beta 2 integrin-dependent adhesion to a not yet identified counter-receptor constitutively expressed on platelet surface.