Beyond Lipoprotein Receptors: Learning from Receptor Knockouts Mouse Models about New Targets for Reduction of the Atherosclerotic Plaque.

Atherosclerosis and its complications represent the leading death cause worldwide, despite many therapeutic developments. Atherosclerosis is a complex, multistage disease whereby perturbed lipid metabolism leads to cholesterol accumulation into the vascular walls and plaque formation. Generation of apoE-/- and LDLR-/- atherosclerosis mouse models opened the avenue for investigating the mechanisms of action for specific molecules. We focus herein on the involvement of non-lipoprotein receptors in atherogenesis, as revealed by their total or site-specific ablation in the aforementioned murine models. The receptors reviewed span a broad range, from molecules related to lipid metabolism (adiponectin receptors) to molecules whose connection with atherogenesis is less obvious (cannabinoid receptors). We also outline cross-transplantation studies which allowed uncoupling the lipid modulating effects from the inflammatory ones. For certain receptors, since knockouts were unavailable, pharmacological data are presented instead. We emphasize the contribution of the receptors to the pathology, based on functional criteria, such as oxidative stress, immune response, inflammation, angiogenesis. Controversial aspects regarding the pro- or anti- atherogenic activity of some receptors are highlighted. We assume these discrepancies are due to the experimental setup, animal models used, tissue-specific action, various isoforms analyzed, divergent signaling or cross-talk between metabolic and immune pathways. Understanding the influences of cellular receptors in the progression of atherosclerosis allows their modulation towards an antiatherogenic phenotype. The experimental studies in animal models were in some cases successfully extrapolated to humans leading to atheroma reduction, and we expect this to occur even to a greater extent, based on the newest achievements.

Regulation of NADPH oxidase subunit p22(phox) by NF-kB in human aortic smooth muscle cells.

Accumulating evidence demonstrates the involvement of oxidative stress in the pathophysiology of cardiovascular diseases. The molecular mechanisms accountable for the increased production of reactive oxygen species remain uncertain. Among others, NADPH oxidase is one of the most important sources of superoxide in vascular cells. Here we investigate the role of NF-kB in the regulation of p22(phox) subunit and NADPH oxidase activity, in human aortic smooth muscle cells. Overexpression of p65/RelA or IKKbeta up-regulated p22(phox) gene promoter activity. Transcription factor pull-down assays demonstrated the physical interaction of p65/RelA protein with predicted NF-kB binding sites. Real time PCR and Western blotting analysis showed that p22(phox) mRNA and protein expression are significantly down-regulated by NF-kB decoy oligodeoxynucleotides and N-alpha-tosyl-l-phenylalanine chloromethyl ketone (TPCK). Lucigenin-enhanced chemiluminescence assay revealed that NF-kB inhibitors reduce the NADPH-dependent superoxide production. Regulation of NADPH oxidase by NF-kB may represent a possible mechanism whereby pro-inflammatory factors induce oxidative stress in atherosclerosis, hypertension, diabetes, stroke or heart failure.

Proliferation, differentiation and characterization of osteoblasts from human BM mesenchymal cells.

BACKGROUND: The objective of this study was to isolate osteoprogenitor cells (OPC) from BM mesenchymal stromal cells (MSC) and test their capacity to proliferate and differentiate into osteoblasts. METHODS: Human MSC were separated on a Percoll gradient and cultured in DMEM supplemented with 15% human serum, and characterized by flow cytometric analyzes for CD34, CD13, CD90, CD105 and CD117. To induce differentiation, cultured cells were exposed to 10(-7) m dexamethasone (dexa) and/or 10(-3) m sodium beta-glycerophosphate (beta-GlyP) and 1,25-dihydroxyvitamin D3 (calcitriol) or 9-cis-retinoic acid (9-RA). RESULTS: alkaline phosphatase (AP) activity was detected in cells irrespective of the dexa and/or beta-GlyP treatment. Antigenic phenotypes of MSC were CD34- (more than 99%) and CD13+ CD90+ CD105+ CD117+ (c. 50%). The treatment induced extracellular calcium deposition and gene and protein expression of osteonectin (ON) and bone sialoprotein (BSP): beta-GlyP induced an increase (c. 2.2-fold) of the ON gene and dexa augmented (c. 2.7-fold) the gene expression of BSP II. Gene expression of BSP I reached a maximum at 3 weeks of combined treatment. Osteocalcin gene expression was induced only after additional treatment with calcitriol or 9-RA. Ultrastructural analysis revealed the secretory phenotype of OPC. DISCUSSION: Under appropriate treatment, MSC can give rise to OPC that have the capacity to differentiate into osteoblasts characterized by the expression of osteogenic markers, osteoblastic properties and stromal BM cells phenotypes. These cells may represent a promising material to be utilized in orthopedic cellular therapy.

Investigation of IgG receptors expressed on the surface of human placental endothelial cells.

Fetal passive immunity is acquired by transfer of maternal IgG through the placental syncytiotrophoblast and endothelium; few and contradictory data exist for IgG transcytosis in human placental endothelial cells (HPEC). In this study, we tested the binding and internalization of IgG by cultured HPEC and the expression of FcgammaRs. Biochemical analysis and microscopy revealed that the binding of IgG occurred through the Fc portion of the molecule and was greater on the basolateral than on the apical cell surface. IgG binding and internalization were saturable and the data calculated from Scatchard plot for IgG surface binding indicated a single interaction with an apparent K(d)of 2x10(-7)M. During 3 h of chase, approximately 10 per cent of IgG was released in an intact form in the medium. By electron microscopy, IgG was detected on HPEC surface, internalized in endothelial caveolae and within endosomal compartments. RT-PCR, blotting and microscopy failed to detect the presence of FcgammaRI-III in HPEC. However, the specific radioiodination and affinity chromatography revealed the presence of a 55 kDa-IgG binding polypeptide on cell surface. These findings indicate that HPEC (i) take up and internalize IgG via a receptor mediated process; (ii) bind IgG prevalently on the basolateral surface via the Fc fragment of the molecule and (iii) exhibit a novel FcgammaR of 55 kDa on the cell membrane.

Severity of oxidative stress generates different mechanisms of endothelial cell death.

The role of reactive oxygen species (ROS) in the pathogenesis of vascular diseases is well established, but few data exist on the mechanisms by which ROS induce endothelial cell (EC) death. We examined the conditions and the mechanisms by which oxidative stress induces EC death, using cultured confluent bovine aortic ECs exposed for 30 min to different concentrations of hydroxyl radicals (HO*) generated by hydrogen peroxide (H(2)O(2)) in the presence of 100 microM ferrous sulfate (FeSO(4)). Cell viability assays, Hoechst DNA staining, TUNEL (TDT-mediated dUTP-biotin nick end-labeling) analysis, agarose gel electrophoresis and annexin V assay were used to determine the effect of HO* on the viability of ECs, and to distinguish between apoptosis and necrosis. The results showed that at concentrations of up to 0.1 mM H(2)O(2)/FeSO(4), the large majority of cells are viable, except for approximately 12.5% death, which occurs by apoptosis. At a concentration of 0.2 mM H(2)O(2), the cell viability is reduced to 66%, while EC apoptosis remained at comparable values (14%). At high oxidative stress (0.5 mM H(2)O(2)), the cell viability was drastically reduced (approximately 39%), and the prevalent form of death was necrosis; apoptosis accounted for only approximately 17%. Together, these data indicate that: (1) HO* induce EC death either by apoptosis or necrosis and (2) the mechanisms of EC death differ as a function of the concentration of HO. Thus, the same insult can cause apoptosis and/or necrosis, as a function of the intensity rather than the nature of the insult.

Expression of functionally active FcRn and the differentiated bidirectional transport of IgG in human placental endothelial cells.

The mechanism of selective transport of the immunoglobulins G from the placental stroma to the lumen of the fetal blood vessels has not been elucidated yet. It was postulated that the specific transport as well as the regulation of IgG level in the blood, involves the MHC class I related receptor FcRn for the Fc domain of IgG. We questioned whether human placental endothelial cells (HPEC) express FcRn and, if present, whether it is in a functionally active form. The experiments were performed on cultured HPEC and as positive control, human trophoblastic (JEG3) and mouse endothelial cells (SVEC) were used. Expression of FcRn, was demonstrated by indirect immunofluorescence and RT-PCR. The role of FcRn was assessed by quantifying the transcellular transport of [(125)I]-hIgG or [(125)I]-rF(ab')(2) fragments from the apical to basolateral surface, and in the reverse direction of HPEC grown on filters in a double chamber system. The intracellular pathway of FcRn or IgG was examined by electron microscopy using the proteins adsorbed to 5 nm and 20 nm colloidal gold particles, respectively. The results showed that: (a) FcRn is expressed by human placental endothelial cells, in a functionally active form; (b) transcytosis of IgG in HPEC is a time-dependent process that takes place preferentially from the basolateral to the apical compartment; and (c) both IgG and FcRn colocalize in an intracellular endocytic compartment, chloroquine sensitive. Together these data suggest that the regulation of IgG level by endothelial cells may result from interplay between salvaging, exocytosis, and transcytosis of the molecules. One can assume that IgG that does not bind to FcRn may be destined for destruction, and this would explain the mechanism by which IgG homeostasis is maintained.

Establishment of a pure vascular endothelial cell line from human placenta.

UNLABELLED: Endothelial cells (EC) from various sectors of the circulatory system have distinct characteristics, some of which have only been identified in cultures upon their isolation from specific organs or tissues. Cultured vascular EC, derived from the human placenta (HPEC), may be helpful for studying their specific function in the fetoplacental unit, such as in the control of maternofetal traffic. In this paper we report an improved method for isolation, purification and culture of HPEC, that implies an enzymatic perfusion of the term placenta, followed by separation of resulting cells on a Percoll density gradient. The inoculated starting suspension was purified by a two-step selection procedure, based on differential trypsinization, leading to a pure population of about 8x10(7)cells/placenta, with 2.7-3.4 population doublings. The average population doubling time during eight passages was 60-65 h and the life span of HPEC was approximately 45-50 population doublings. The cell morphology at optical and electron microscopical level revealed a good differentiation of HPEC, which were endowed with numerous plasmalemmal vesicles (caveolae) and Weibel-Palade bodies. The transendothelial electrical resistance of the HPEC monolayer varied between 22 and 52 Ohm/cm(2). The cultures were mycoplasma free, as revealed by fluorescence microscopy using DNA dyes and the polymerase chain reaction (PCR). The negative immunofluorescent reaction for keratin confirmed that the HPEC were not contaminated with either type of placenta cells, as syncytiotrophoblast. Cultured HPEC demonstrated a strong reaction for von Willebrand factor antigen (by fluorescence microscopy), took up AcLDL-DiI and expressed active angiotensin converting enzyme. These characteristics substantiate the endothelial nature of cultured cells. The interactions with different lectins (BS-I, SBA, RCA, UEA and WGA) assessed by fluorescence microscopy and blotting reveal a strong reaction of HPEC with UEA and a negligible reaction with BS-I lectin. WGA lectin displayed a marked fluorescence staining in subconfluent HPEC, and at the level of intracellular clefts in post-confluent cultures. IN CONCLUSION: (i) we have obtained a pure line of cultured EC originating from the human placental venous side of the circulatory tree; (ii) the cells have the general characteristics and markers ascribed to EC; (iii) as opposed to large human placental vessels, HPEC do not react to BS-I lectin and, unlike human umbilical vein EC, have a much higher proliferation rate and a long lifespan; (iv) HPEC expressed a characteristic glycosylated coat particularly rich in alpha- L -fucose and beta-GlcNAc containing glycocompounds.

Protein and fatty acid composition of caveolae from apical plasmalemma of aortic endothelial cells.

In endothelial cells (EC), caveolae or plasmalemmal vesicles (PVs) represent a structurally and biochemically specialized membrane microdomain. Since few data are available on the biochemical composition of PVs of large vessel endothelium, we have designed experiments to isolate this domain and to analyze its chemical components. A highly purified apical membrane fraction was obtained from cultured bovine aortic EC by using cationic colloidal silica (silica-ap), or the EC were surface-radioiodinated and a cell homogenate was prepared. Detergent treatment (Triton X-100; TX) and mechanical disruption of both the silica-ap fraction and cell homogenate followed by ultracentrifugation on a sucrose gradient gave detergent-soluble and detergent-insoluble membranous fractions. The lowest density TX-insoluble fraction appeared morphologically as distinct vesicles (caveolae; 60 nm average diameter; PVs fraction). Biochemical characterization of the PVs fraction (by comparison with the soluble fraction) revealed the presence, at high concentration, of specific caveolar markers, viz., caveolin (both isoforms, the 24-kDa form being conspicuously more abundant) and Ca2+-ATPase. By contrast, angiotensin-converting enzyme and alkaline phosphodiesterase were present almost exclusively in the TX-soluble fraction. The glycoproteins in the PVs fraction were of apparent molecular weights 52, 68, 95, and 114 kDa. Analysis of the fatty acid composition revealed more palmitoleic and stearic acid in the PVs fraction then in the TX-soluble fraction. Thus, in comparison with the plasmalemma proper, the PVs fraction (1) is detergent-insoluble; (2) contains caveolin in two isoforms; (3) contains Ca2+-ATPase at high concentration; (4) contains a set of specific glycoproteins; and (5) is enriched in palmitoleic and stearic acids.

Expression of transferrin receptors in endothelial cells transfected by electroporation.

Transferrin is the primary iron-binding protein in the plasma. Transferrin receptors (TfR) were detected in brain and liver endothelial cells (EC); however, little information exists about their intracellular routes. To detect the EC structures involved in TfR biosynthetic and endocytotic pathways, cultured aortic EC were transfected with the plasmid pSR alpha containing a construct encoding the human TfR, to which horseradish peroxidase (HRP) was anchored as reporter molecule. Since EC are difficult to be transfected, we tried different techniques, and two forms of the plasmid (circular and linearized), of which the electroporation method and the linearized plasmid were the most efficient in producing stable cell lines. Transfected cells were selected with geneticin, and the expression of TfR-HRP tested by cytochemistry. The stable transformants preserved the general characteristics of EC. At the ultrastructural level, TfR-HRP was associated with the nuclear envelope, rough endoplasmic reticulum, Golgi complex and adjacent secretory vesicles, cytoplasmic vesicles of various sizes (50-130 nm diameter), endosomes, plasma membrane, plasmalemmal pits, and a fraction of plasmalemmal vesicles. The intensity of the reaction product varied, suggesting a different concentration of TfR, in specific organelles. For example, (i) a gradient of HRP-reaction product was found within the Golgi cisternae, (ii) the plasmalemmal pits were more intensely stained than the adjacent plasma membrane, and (iii) the vesicle membrane was decorated stronger than the endosomal membrane (to which it fuses). A striking feature was the coexistence within the same EC of two vesicle populations (or subtypes): some containing TfR-HRP, whereas others lack the receptor. Quantitative data indicated a stronger expression of TfR in confluent cells (approximately 8-fold higher) than in EC at 2 days after plating; a significant decrease (approximately 9-fold) of TfR was found in postconfluent transfectants. Together, the data demonstrate that (i) after electroporation of EC, the stable lines maintain the characteristics of native cells; (ii) the newly synthesized TfR is located in variable concentration within the organelles involved in endocytosis and exocytosis, and (iii) the expression of TfR-HRP is particularly high in confluent cells.

[Lithium carbonate (Contemnol--Spofa). A clinical study]. / Carbonatul de litiu (Contemnol--Spofa). Studiu clinic.

The estimation of the present day stage in the therapy with lithium salts allowed the authors some considerations on the mechanisms of action and pharmacodynamics of lithium ion, as well as on the indications and counterindications, side effects and therapeutical management. Thus, the authors have analysed the findings of a simple-blind clinical study on lithium carbonate (Contemnol--Spofa) in a series of 15 inpatients. The research methodology included the thorough examination of the patients, blood lithium screening, psychological investigations and laboratory tests on the basis of an itemized investigation card. The analysis of the results confirmed the efficacy of this substance in bipolar hyperthymic psychosyndromes, depressions and hyperthymic psychosyndromes with dissociative elements, its tolerance and low incidence of side effects. The selection of the cases, the clinical and paraclinical examinations, the observance of the indications and counterindications permit the administration of this substance as a curative and prophylactic treatment, providing the blood lithium is screened and clinical control and biological investigations are carried out.