Laser deposition of poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) - lysozyme microspheres based coatings with anti-microbial properties.

The purpose of this study was to obtain, characterize and evaluate the cytotoxicity and antimicrobial activity of coatings based on poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) - Lysozyme (P(3HB-3HV)/Lys) and P(3HB-3HV) - Polyethylene glycol - Lysozyme (P(3HB-3HV)/PEG/Lys) spheres prepared by Matrix Assisted Pulsed Laser Evaporation (MAPLE) technique, in order to obtain functional and improved Ti-based implants. Morphological investigation of the coatings by Infrared Microscopy (IRM) and SEM revealed that the average diameter of P(3HB-3HV)/Lys spheres is around 2µm and unlike the drop cast samples, IRM recorded on MAPLE films revealed a good distribution of monitored functional groups on the entire scanned surface. The biological evaluation of MAPLE structured surfaces revealed an improved biocompatibility with respect to osteoblasts and endothelial cells as compared with Ti substrates and an enhanced anti-biofilm effect against Gram positive (Staphylococcus aureus) and Gram negative (Pseudomonas aeruginosa) tested strains. Thus, we propose that the fabricated P(3HB-3HV)/PEG/Lys and P(3HB-3HV)/Lys microspheres may be efficiently used as a matrix for controlled local drug delivery, with practical applications in developing improved medical surfaces for the reduction of implant-associated infections.

Functional ultrastructure of the vascular endothelium: changes in various pathologies.

Biology has revealed that form follows function or function creates the organ. Translating this law at the cellular level, we may say that the ultrastructure follows function or function creates the ultrastructure. The vascular endothelium is an accurate illustration of this rule due to its numerous and many-sided functions carried out by highly specialised cells, structurally equipped for their tasks. Occupying a strategic position between the blood and tissues, the endothelial cell (EC) tightly monitors the transport of plasma molecules, employing bidirectional receptor-mediated and receptor-independent transcytosis and endocytosis, regulates the vascular tone, synthesises and secretes a large variety of factors, and is implicated in the regulation of cell cholesterol, lipid homeostasis, signal transduction, immunity, inflammation and haemostasis. Ultrastructurally, besides the common set of organelles, the characteristic features of the ECs are the particularly high number of vesicles (caveolae) endowed with numerous receptors, transendothelial channels, the specialised plasma membrane microdomains of distinct chemistry, and characteristic intercellular junctions. In addition, by virtue of their number (-6 x 10(13)), aggregated mass (-1 kg), large surface area (-7,000 m2) and distribution throughout the body, the ECs can perform all the assumed functions. The vascular endothelium, with its broad spectrum of paracrine, endocrine and autocrine functions, can be regarded as a multifunctional organ and chief governor of body homeostasis. The ECs exists in a high-risk position. The cells react progressively to aggressive factors, at first by modulation of the constitutive functions (permeability, synthesis), followed by EC dysfunction (loss, impairment or new functions); if the insults persist (in time or intensity), cell damage and death ultimately occur. In conclusion, the ECs are daring cells that have the functional-structural attributes to adapt to the ever-changing surrounding milieu, to use innate mechanisms to confront and defend against insults and to monitor and maintain the body's homeostasis.

The MHC class I-related receptor, FcRn, plays an essential role in the maternofetal transfer of gamma-globulin in humans.

The transfer of maternal gamma-globulin (IgG) provides the neonate with humoral immunity during early life. In humans, maternal IgG is transported across the placenta during the third trimester of pregnancy. The expression of the MHC class I-related receptor, FcRn, in the human placenta suggests that this Fc receptor might be involved in the delivery of maternal IgG, but direct evidence to support this is lacking. In the current study an ex vivo placental model has been used to analyze the maternofetal transfer of a recombinant, humanized (IgG1) antibody in which His435 has been mutated to alanine (H435A). In vitro binding studies using surface plasmon resonance indicate that the mutation ablates binding of the antibody to recombinant mouse and human FcRn. Relative to the wild-type antibody, the H435A mutant is deficient in transfer across the placenta. Significantly, the mutation does not affect binding to Fc gamma RIII, an FcR that has been suggested in earlier studies to mediate the transfer of maternal IgG. The analyses demonstrate that binding of an IgG to FcRn is a prerequisite for transport across the perfused placenta. FcRn therefore plays a central role in the maternofetal delivery of IgG and this has implications for the use of protein engineering to improve the properties of therapeutic antibodies.

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.

Low density lipoprotein binding induces asymmetric redistribution of the low density lipoprotein receptors in endothelial cells.

The uptake and transport of cholesterol-carrying low density lipoprotein (LDL) by the arterial wall is a continuous dynamic process, contributing to the cholesterol homeostasis in the plasma and in the cellular components of the vessel wall. Upon exposure to endothelial cells (EC), LDL interacts in part, with specific surface receptors (LDL-R). In this study we questioned: (i) the distribution of LDL receptors on the apical and basal cell membranes in endothelial cells; (ii) the role of LDL receptors in the control of cholesterol homeostasis and (iii) the translocation of LDL receptor across the EC. To this purpose bovine aortic EC were cultured on filters in a double-chamber system, in Dulbecco's medium supplemented either with 10% fetal calf serum (FCS) or with 10% lipoprotein-deficient serum (LPDS). The cells were exposed for 3h to 13H]acetate (40 microCi) added to both compartments of the cell culture inserts. The newly synthesized [3H]cholesterol was detected by thin layer chromatography and quantified by liquid scintillation counting. The LDL-R were detected in EC protein homogenates by immunoblotting using a monoclonal antibody against LDL-R (IgG-C7); the intracellular pathway of LDL-R was examined by electron microscopy using a complex made of protein A 5 nm or 20 nm colloidal gold particles and an anti-LDL receptor antibody (Au-PA-C7). To evaluate the distribution and the transport of LDL-R from one cell surface to the other, EC grown in LPDS were radioiodinated either on the apical or on the basolateral surface, incubated on the same surface with LDL, and subsequently biotinylated on the opposite non-radiolabeled surface. The EC were further solubilized and the protein extract immunoprecipitated with anti-LDL-R antibody or with mouse IgG (as control). The eluted antigen-antibody complexes were precipitated with streptavidin-agarose beads, solubilized, and subjected to SDS-PAGE. The results showed that: (a) the LDL-R were present on both endothelial cell fronts; (b) using the complex Au-PA-C7, the LDL-R were localized in endothelial plasmalemmal vesicles as well as coated pits and coated vesicles in multivesicular bodies and lysosomes, irrespective of the cell surface exposed to the complex; (c) biochemical assays indicated that upon ligand binding, the LDL-R were translocated preferentially from the apical to the basal plasma membrane.

Functional expression of the MHC class I-related receptor, FcRn, in endothelial cells of mice.

Our recent data indicate that the MHC class I-related receptor, FcRn, plays a role in regulating serum IgG levels, in addition to its known role in transferring IgG from mother to young. In the current study, the distribution of FcRn in adult mice has been investigated using several approaches. First, tissue distribution of anti-FcRn F(ab')2, murine IgG1 and recombinant, IgG1-derived Fc-hinge fragments has been analyzed, and these FcRn binding proteins localize predominantly in skin and muscle with lesser amounts in liver and adipose tissue. Second, histochemical analyses of muscle and liver with anti-FcRn F(ab')2 indicate that FcRn is expressed in the endothelium of small arterioles and capillaries, but not in larger vessels such as the central vein and portal vasculature. Third, immunoprecipitation and immunofluorescence studies of cultured murine endothelial cells show that functional FcRn is expressed in these cells, and is located within vesicular structures in the cytosol and not on the membrane. Taken together the data demonstrate that FcRn is expressed in functionally active form in endothelial cells, indicating that these cells are a possible site at which serum IgG homeostasis is maintained.

Heart microvessels and aortic endothelial cells express the 15 kDa heart-type fatty acid-binding proteins.

Due to their hydrophobic nature, free fatty acids require carriers for transport across and within the cells. The endothelial layer is the first barrier to be traversed by the fatty acids, from the plasma to the underlying cells and tissues. We tried to find out whether cytosolic fatty acid-binding proteins (FABPs) are present in the endothelium of large vessels (aortic endothelial cells) and small vessels (myocardial capillaries) using the following experimental approaches: (i) loading the delipidated aortic endothelial cell (EC) homogenate and the heart cytosolic proteins and membrane proteins with [14C]palmitate or [14C]oleate, respectively, followed by autoradiographic detection of electrophoretically separated bands; (ii) detection by immunoprecipitation of heart-type FABP (H-FABP) using an affinity-purified antibody raised against bovine H-FABP (anti-H-FABP), and (iii) localization of FABP by indirect immunofluorescence and gold-immunocytochemistry applied to cultured EC and to thick and thin frozen sections of mouse heart. The results showed that: (i) within the EC homogenate proteins that express affinity for [14C]palmitate have an apparent Mr of 15000, and 40000-45000, that correspond as molecular mass to cytosolic and membrane FABPs, respectively. Similar affinity was found by incubation with [14C]oleate, that binds to a protein of Mr 15000 in the heart cytosol, and to a 40-45 kDa protein in the membrane fraction; (ii) anti-H-FABP immunoprecipitated specifically a cytosolic 15 kDa peptide (H-FABP); (iii) by indirect immunofluorescence, cytosolic H-FABP was localized on heart microvessels and myocytes and also in cultured aortic EC where intense spotted fluorescence characteristic for cytosolic antigens was present; (iv) by immunocytochemistry, H-FABP was detected in the EC cytoplasm, and in close proximity to the cytoplasmic aspect of plasmalemma and vesicle membranes. Together the data attest the presence of the 15 kDa, heart-type FABP in the endothelium of aorta and heart microvessels.

Transcytosis of albumin in endothelial cells is brefeldin A--independent.

To determine whether in endothelial cells (EC) the pathways of endocytosis and transcytosis of macromolecules interconnect, the effect of Brefeldin A (BFA) on these processes was tested. To this purpose EC were grown to confluence on plastic culture dishes or on cell culture chamber inserts placed into corresponding wells, so as to obtain a dual chamber system. The cells maintained the typical characteristics of EC and had an electrical resistance in the range of 30-60 Ohm.cm2. Transendothelial transport of albumin conjugated to the fluorochrom Texas Red (Alb-TR) and of horseradish peroxidase (HRP) added to the upper compartment, in the absence or presence of BFA (0-25 micrograms/ml), was evaluated in aliquots collected from the lower compartment. At different time intervals, quantitative data were obtained by fluorimetry and spectrophotometry. In other experiments transcytosis of Alb-TR was examined in the presence of 100 microM forskolin (an inhibitor of BFA effect). The endocytosis of Alb-TR and HRP was evaluated by incubating EC with the probes, and the internalized tracers determined in the cell lysate using the methods described above. The results showed that BFA has no significant effect on transcytosis of albumin and HRP. In contradistinction, BFA (5 micrograms/ml) reduced markedly endocytosis of HRP (by 47%). Forskolin has no effect on transcytosis. The data indicate that the BFA-induced perturbance in the endocytic route does not affect the transcytotic pathway of albumin, and suggest that in EC, transcytosis of macromolecules may represent a shortcut for rapid and direct transport of some plasma molecules across the cell.

A method for selective radiolabeling of lung endothelium plasmalemmal vesicles, in situ.

Albumin-gold complex (Alb-Au) was previously shown to bind selectively to plasmalemmal vesicles of capillary endothelium. Based on these findings, as well as on the ability of lactoperoxidase (LPO) to mediate the radioiodination of proteins, we have prepared a complex of gold particles bearing both albumin and anionized lactoperoxidase (Alb-Au-aLPO). The complex had a pI of 5.8, largely preserved aLPO enzymatic activity (approximately 74%), and was able to catalyze protein radioiodination. Upon washing out the blood, the complex was perfused in the mouse lung, the excess tracer removed, and a Na125I/H2O2 solution was introduced in the vasculature. After extensive washing, lung fragments were processed for either electron microscopy (EM), or to prepare a membrane-enriched fraction. In control experiments, lungs were perfused with native LPO (pI 9.3), or with a LPO-Affi Gel conjugate and further radioiodinated as described for Alb-Au-aLPO. By EM, it was found that both in tissue and in the isolated membrane fraction, only Alb-Au-aLPO labeled markedly and preferentially some uncoated pits and most plasmalemmal vesicles. Analysis by SDS-PAGE and autoradiography of a membrane-enriched fraction prepared from lungs perfused with Alb-Au-aLPO had some major identified 125I-labeled polypeptides of apparent molecular masses of 16, 18, 31, 36, 55, and 77 kDa. A different subset of polypeptides was labeled in lungs perfused with LPO, whereas after administration of LPO-Affi Gel the major radiolabeled polypeptides had a molecular mass of 33, 55 kDa and several peptides in the range of 77 to 160 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

Albumin-binding proteins function in the receptor-mediated binding and transcytosis of albumin across cultured endothelial cells.

The functional significance of the endothelial albumin-binding proteins (ABP) was tested on cultured bovine aortic endothelial cells (BAEC) incubated with radiolabeled albumin ([125I]Alb) alone or carrying fatty acids (oleic acid (OA) or arachidonic acid (AA)) or triiodothyronine. The [125I]Alb binding was estimated on BAEC grown on 96-well plates, and the transport was evaluated on BAEC cultured in a dual chamber system. The probe interaction with the monolayer was monitored as a function of concentration and temperature in the presence or absence of either unlabeled Alb or an anti-albumin anti-idiotypic antibody (Ab2), which was previously demonstrated to specifically recognize the ABP of endothelial cell surface. Cultured BAEC bound specifically and with high affinity [125I]Alb. The binding of fluorescein isothiocyanate (FITC)-Alb to endothelial cells was inhibited by Ab2 in immunofluorescence studies. As compared to albumin, the binding of albumin carrying either OA or AA was higher and was diminished by Ab2. Transport of [125I]Alb across BAEC grown on gelatin-coated filters increased with time, and after 60 min, approximately 30% of [125I]Alb was transported from the upper to the lower compartment; unlabeled Alb or Ab2 reduced this process by approximately 75%. Colchicine decreased transcytosis of [125I]Alb by approximately 80%, whereas chloroquine by approximately 27%. Transendothelial transport of [125I]Alb carrying fatty acids was 40% and 20% higher for OA and AA, respectively, as compared to that of defatted albumin. The results suggest the coexistence of a receptor-mediated and a receptor-independent transcytosis of albumin across cultured endothelial cells; ABP of the endothelial cell surface appear to be involved in the specific binding and transport of albumin.

Albumin binding proteins of endothelial cells identified by anti-idiotypic antibodies.

Studies were conducted to identify and localize albumin binding proteins (ABPs) in endothelial cells using rabbits polyclonal anti-idiotypic antibodies (Ab2) raised against the affinity-purified anti-bovine serum albumin IgG. Ab2 were purified by fast performance liquid chromatography. The anti-idiotypic nature of the IgG was assessed by (i) the capacity to inhibit albumin binding to its specific antibody in a dose-dependent manner, (ii) the lack of interaction with albumin, and (iii) the interaction with anti-albumin antibodies of diverse origins. The latter characteristic indicates that although polyclonal, the purified anti-idiotypic antibodies contain some of the Ab2 beta type. The binding of Ab2 to cultured bovine aortic endothelial cell surfaces was saturable and specific as demonstrated by radioimmunoassay (RIA) and immunofluorescence studies respectively. A competitive RIA was used to test whether Ab2 competed for albumin binding to bovine aortic endothelial cells (BAECs) (presumably to ABPs). It was found that Ab2 inhibited binding of [125I]albumin to BAECs in a dose-dependent fashion. Immunoblot analysis of extracts of BAECs, microvascular endothelial cells, and lung showed that both Ab2 and albumin bind specifically to two polypeptides with an apparent molecular mass of 18 and 31 kDa. In addition, upon radioiodination of BAECs apical membrane proteins, Ab2 bound specifically and immunoprecipitated restrictively two radiolabeled cell surface proteins of 18 and 31 kDa. The results provide direct evidence for the presence of the 18 and 31 kDa peptides (ABPs) on the endothelial cell membrane and/or associated structures, i.e. open plasmalemmal vesicles and uncoated pits.

Albumin-binding proteins of endothelial cells: immunocytochemical detection of the 18 kDa peptide.

Extracts of isolated microvascular endothelial cells (MEC) and cultured bovine aortic endothelial cells (BAEC) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), electrotransfer and incubation with albumin either radioiodinated or adsorbed to 5-nm gold particles. Both ligands reacted exclusively with two peptides of 18 and 31 kDa. To the 18 kDa peptide (excised from preparative SDS-PAGE), an antibody was raised in rabbits and purified by affinity on 18 kDa obtained from two-dimensional gel electrophoresis and immobilized on nitrocellulose paper. The specificity of the anti-18 kDa was assessed by immunoblotting and immunoprecipitation of endothelial cell extracts. To check whether the 18 kDa peptide is exposed on the endothelial cell surface and/or its components (uncoated pits, open plasmalemmal vesicles), the apical membrane of BAEC was radioiodinated, the solubilized proteins incubated with the anti-18 kDa, and the immune complexes formed were precipitated with protein A-Sepharose CL-4B. The ensuing SDS-PAGE and autoradiography revealed that from all radioiodinatable surface proteins, the 18 kDa was the only polypeptide immunoprecipitated by the anti-18 kDa antibody. To localize the 18 kDa peptide, we applied indirect immunofluorescence technique on cultured MEC and BAEC and immunoelectron microscopy (EM) on ultrathin cryosections of mouse heart. Nonpermeabilized whole MEC and BAEC incubated with anti-18 kDa followed by rhodamine-conjugated second antibody showed a relatively intense surface fluorescence often appearing as small dots. At the EM level, heart ultrathin cryosections exposed anti-18 kDa followed by gold-conjugated second antibody revealed that 18 kDa was primarily associated with the membrane of plasmalemmal vesicles of capillary endothelia.(ABSTRACT TRUNCATED AT 250 WORDS)

An ultrastructural study of beta-very low density lipoprotein uptake and transport by valvular endothelium of hyperlipidemic rabbits.

The uptake and transport of beta-VLDL by the aortic valvular endothelium of normal and hypercholesterolemic rabbits were studied in situ. The rabbits were fed either standard diet or standard diet supplemented with 0.5% cholesterol and 10% butter, for 1-20 weeks. Rabbit beta-VLDL was either radiolabeled with 125I or conjugated with colloidal gold (Au) (5 nm-diameter size). beta-VLDL-Au or [125I]-beta-VLDL was perfused in situ via left ventricle and maintained within heart and thoracic aorta for 15 to 60 min at 37 degrees C. Small pieces of aortic valve tissue were processed for electron microscopy or for electron microscopic autoradiography according to the lipoprotein probe used. beta-VLDL was taken and transported by the valvular endothelium of normal or hypercholesterolemic animals by endocytosis and transcytosis. As compared to normal animals, the endocytosis of beta-VLDL by the aortic valvular endothelium of hypercholesterolemic animals started to increase after 2-3 weeks of diet, being almost double in the 12th week of diet. Quantification of autoradiographic grains indicated a marked augmentation of beta-VLDL particles transcytosed by plasmalemmal vesicles of hypercholesterolemic animals as compared to normal. In the aortic valves of hyperlipidemic animals, the amount of beta-VLDL transported through the endothelium of fibrosa region was 3 times higher than that of spongiosa region of the same valve. The results showed that in hyperlipidemia, beta-VLDL transcytosis is generally markedly enhanced, in particular in lesion prone area.

Evidence for thyroxine transport by the lung and heart capillary endothelium.

The uptake and transport of carrier-bound thyroxine by the endothelium were investigated by perfusing through the heart and lung of young rats radiolabeled thyroxine bound to prealbumin ([125I]T4Pa) or serum ([125I]T4S). In addition these complexes were tagged to 5-nm gold particles to obtain quantitative (radioassay) and qualitative (autoradiography) data from the same experiment. The complexes (prewarmed at 37 degrees) were perfused in situ at various concentrations (1 to 50 muCi/ml) for time intervals ranging from 5 to 30 min. After thorough washing of the unbound probe, tissue fragments were either measured for total uptake in a gamma counter or processed for electron microscopy autoradiography. The results showed that both the lung and heart take up [125I]T4 complexes by a process that is saturable at low hormone concentration; uptake is completed by free T4 and Pa. In specimens perfused with double-labeled complexes (iodinated and tagged to gold) autoradiography revealed that silver grains and gold particles colocalize predominantly on endothelial plasmalemmal vesicles. The probe appeared first in vesicles open to the capillary lumen (5 min) and further on in vesicles apparently free within the cytoplasm or open to the abluminal front. At 30 min, only silver grains seem to be present in the pericapillary space, on the alveolar epithelial cells, as well as on the nucleus and mitochondria of heart myocytes. The findings suggest that (1) T4Pa uptake by the endothelial cell (EC) is a specific process (possibly via specific binding sites); (2) T4Pa is taken up and transported across capillary EC by plasmalemmal vesicles; (3) in the pericapillary space T4 seems to dissociate from its carrier.

Transport pathways of beta-VLDL by aortic endothelium of normal and hypercholesterolemic rabbits.

The uptake and transport of beta-VLDL by the aortic endothelium was investigated in normal and hyperlipidemic rabbits fed a cholesterol-enriched diet for 1 week to 5 months. Weekly (in the first month) or every other week afterwards, animals were given one of the following probes: (a) [125I]-beta-VLDL injected in vivo and after 24 h the whole aorta or its intima and media were separately collected and examined by spectrometry and autoradiography; (b) [125I]-beta-VLDL coupled to the fluorescent probe 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate perfused in situ for 1-2 h and aorta examined by radioassay and fluorescence microscopy; (c) beta-VLDL-gold complex perfused in situ for 10-15 min and aortic fragments examined by electron microscopy. In addition, cryosections of aortic wall were processed for the immunocytochemical detection of apolipoprotein B and apolipoprotein E. The results showed that both in normal and hyperlipidemic rabbits, the aortic endothelium transports plasma beta-VLDL by a dual pathway: (i) endocytosis involving coated pits and vesicles, endosomes, multivesicular bodies and lysosomes, and (ii) transcytosis, the predominant process, carried out by plasmalemmal vesicles. Both processes, and especially transcytosis, are markedly increased in hyperlipidemia leading to progressive accumulation of beta-VLDL or/and its components in the subendothelial extracellular matrix. In prelesional stages of atherogenesis, beta-VLDL-gold complexes or deposits of apo B and apo E were detected in close association with extracellular liposomes. With the appearance of intimal macrophage-derived foam cells, the immunoperoxidase reaction product, revealing the presence of the two apolipoproteins, could also be seen in intracellular lipid inclusions.

Histamine receptor on mast cells.

Mast cells isolated from the peritoneal fluid of Wistar rats were purified by centrifugation on Percoll gradient with a yield of 2x10(6) cells/ml. Cell morphology was well preserved as shown by light and electron microscopy. The mast cells capacity to bind histamine was assayed using either [3H]-histamine or histamine-ferritin conjugate as electron-opaque probe for electron microscopic examination. The [3H]-histamine binding performed at 4 degrees C in Ca2+-free phosphate-buffered saline pH 7.3 completed within 30 min was found to be specific, with an IC50 value of 0.72 +/- 0.23 nM. The data analyses by Scatchard and Hill's representations showed a KD of 0.60 +/- 0.24 nM and Bmax of 4.9 +/- 1.2 pM/10(6) cells suggesting that on mast cells the histamine receptors are restricted to the plasma membrane. According to Hill's analysis neither positive nor negative cooperativity (n = 1.06) appeared to be involved in the specific histamine-receptor binding. Competition experiments with 4-methylhistamine and SK&F 93479, revealed that mast cells express H2-histamine receptors. At electron microscopic level, the histamine-ferritin conjugate interstitially injected in the hamster cheek pouch was localized on the mast cell membrane.

The permeability of aortic endothelium to 125I-BSA in hyperlipidemic hamster. Effect of histamine and serotonin.

The aortic permeability to 125I-bovine serum albumin (BSA) and the effect of histamine and serotonin were investigated in hamsters with diet-induced hyperlipidemia. Animals, fed a cholesterol- and butter-rich diet for five weeks, were sacrificed weekly and the level of serum cholesterol, the aortic permeability, and the morphologic aspect of the vessel wall, were analysed. During this interval, the cholesterolemia increased four-fold, and a progressive lipid accumulation in the intima was recorded. The permeability of the aortic wall was assessed by injecting 125I-BSA into the circulation and detecting the radioactivity in the aortic wall. The effect of the two vasoactive amines was determined by injecting histamine or serotonin concomitantly with the 125I-BSA. The results indicate that during the early stages of experimental hyperlipidemia in hamster, the aortic wall shows an increased permeability to albumin. The process is markedly augmented by histamine and serotonin, predominantly in the abdominal aorta.

Further evidence for the distribution and nature of histamine receptors on microvascular endothelium.

The localization of histamine receptors and their classes on the microvascular endothelium was assessed by using the electron-opaque conjugate histamine-ferritin (HF). In order to further check the specific binding of this conjugate, two compounds chemically similar to histamine but biologically inactive, tele-methylhistamine (t-MH) and 4-pyridylethylamine (PEA), and a specific H2 histamine receptor antagonist, SK&F 93479, were used. Glutaraldehyde-activated ferritin was covalently coupled with either histamine, as a biologically active HF conjugate, or with tele-methylhistamine, as a biologically inactive conjugate tele-methylhistamine-ferritin (t-MHF). The purity of the conjugates was determined by thin-layer chromatography. The HF conjugate (25 mg protein/100 g body weight) was perfused into RAP mice and bipolar microvascular fields of the diaphragm were fixed and processed for electron microscopy. The HF conjugate decorated restricted domains of the luminal aspect of the microvascular endothelium. The specific density of HF binding, recorded as the average number of binding sites per square micrometer of the luminal endothelial surface, was relatively high in venules (18.46 +/- 5.73) and lower in arterioles (12.89 +/- 6.13) and capillaries (9.51 +/- 4.20). The binding specificity of the HF conjugate was assessed through six groups of experiments: perfusion before HF conjugate with either histamine, tele-methylhistamine or 4-pyridylethylamine, perfusion with t-MHF conjugate only, or administration before t-MHF conjugate of either histamine or tele-methylhistamine. The statistical significance of the data was analyzed by using the "F" distribution test and the "Wilcoxon-Mann-Whitney rank-sum test". Both tele-methylhistamine and 4-pyridylethylamine as well as histamine, showed a higher competition on arterioles and venules than on capillaries. Neither tele-methylhistamine nor histamine inhibited the binding of t-MHF conjugate on the endothelium. Experiments using SK&F 93479 as a very potent H2 histamine receptor antagonist perfused before HF administration, suggested the existence of H2 histamine receptors at a higher concentration in venules (47.03%) and arterioles (38.80%) than in capillaries (14.16%). These findings demonstrate that HF conjugate binds specifically to the histamine receptors of microvascular endothelium, which are characteristically frequent and predominantly of H2 type in venules.