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.

The effects of simultaneous hyperlipemia-hyperglycemia on the resistance arteries, myocardium and kidney glomeruli.

The experimental model of Golden Syrian hamster subjected to concomitant hyperlipemia (diet-induced) and diabetes (by streptozotocin injection) for 24 weeks is characterised by the prevalence of micro- and macroangiopathies. We have used the hyperlipemic-diabetic (HD) hamsters to investigate: a) whether there is an alteration in the reactivity of the resistance arteries (mean internal diameter: 210-250 microm), b) if present, which are the structural and biochemical changes that accompany the functional modifications, and c) to examine the pathomorphological changes induced by the association of hyperlipemia and diabetes on vital organs such as myocardium and kidney glomeruli. To these aims, biochemical assays of plasma components, light- and electronmicroscopy, myographic, morphometric and spectrofluorimetric techniques were used. The mesenteric resistance arteries of HD hamsters exhibited (as compared to similar arteries in normals) a decreased contractile response to noradrenaline (1.86+/-0.35 vs. 2.43+/-0.21), and an impeded endothelium dependent relaxation to acetylcholine (approximately 61.40% vs. approximately 79.80%). The association of hyperlipemia with diabetes induced changes in morphology of the resistance arteries consisting in approximately 10% increase of the intima plus media cross-sectional area, approximately 20% decrease of the vascular lumen area, and approximately 2.85 fold augmentation of the wall to lumen ratio. The resistance arteries exhibited structural modifications of the endothelium (up to 8 copies of Weibel-Palade bodies/endothelial cell), and smooth muscle cells (secretory phenotype), and in the vessels media small calcification cores appeared embedded in a hyperplasic extracellular matrix. The vascular mesenteric bed of the HD hamsters contained approximately 2.30 and approximately 1.30 fold increased concentrations of AGE-collagen and pentosidine, respectively, above the normal values. The HD hamsters displayed also modifications that may be dependent on or may lead to an increase in blood pressure, such as: a) approximately 2 fold increase in the activity of serum angiotensin converting enzyme; b) approximately 4.8 fold enhancement of erythrocytes fragility (as a measure of the oxidative stress); c) left ventricular hypertrophy associated with a progressive disarray of cardiomyocyte contractile fibers, interruptions of the Z bands, and accumulation of collagen-rich extracellular matrix indicative of interstitial fibrosis; d) the kidney glomerular capillaries appeared partially or totally collapsed, with a thickened basement membrane which appeared polymorphic, and in some locations made up of successive layers connected by fine bridges and intercalated nodules; in addition, an increase (approximately 1.50 fold) of the mesangial volume was indicative of glomerulosclerosis.

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.

The effects of low density lipoproteins modified by incubation with chondroitin 6-sulfate on human aortic smooth muscle cells.

One of the first changes that take place within the artery intima at the inception of atherosclerosis is the accumulation of LDL-derived modified lipoproteins which appear as subendothelial lipid droplets and vesicles. With time, the LDL retention and interaction with intimal chondroitin sulfate-proteoglycans may induce further structural and functional modification of the lipoproteins. The aim of this study was to produce 'in vitro' modified lipoproteins by LDL incubation with chondroitin 6-sulfate (CS, at 37 degrees C, for 48 h, in the absence of antioxidants) and to test their effects on cultured human aortic smooth muscle cells (SMCs). CS induced LDL modification (CS-mLDL) consisted in formation of a mixture of fused particles (up to 150 nm diameter) and monomers with a small content of lipid peroxides and a partially degraded apo B-100, corresponding to a mild oxidation. Upon incubation with SMCs, CS-mLDL produced a concentration-dependent stimulation of 3H-thymidine incorporation, that, at low concentration (25 microg/ml), was 2-3-fold higher than that obtained when native LDL was used; this increase correlates well with the level of CS-mLDL uptake at the same concentration. Besides the mitogenic effect, CS-mLDL induced a significant stimulation of SMCs migration, comparable with that reported for oxidized LDL. Upon incubation with CS-mLDL, SMCs accumulated lipid droplets of various number and dimension, as revealed by Nile red staining and electron microscopy. Competition studies performed in the presence of 20-fold excess of native LDL and acetyl LDL showed that 125I-CS-mLDL were taken up both by LDL receptor and scavenger receptor. At high concentration (200 microg/ml), CS-mLDL had a cytotoxic effect that was not significantly different from that of native LDL. Together these results provide evidence of (i) the direct alteration produced by CS on LDL and (ii) the effect of CS-mLDL on SMCs migration, proliferation and transformation in lipid-laden cells, events that are crucial in the development of fibro-muscular atherosclerotic lesions.

The interaction between human low density lipoproteins and bovine aortic endothelial cells. Measurements of membrane fluidity.

Bovine aortic endothelial cells in culture have been incubated with human low density lipoproteins (LDL) characterized in their cholesterol content. The incubation was done at different time intervals up to 72 h and various LDL concentrations. It began after endothelial cells had been starved for 24 h in lipoprotein deficient serum. The transfer of some LDL-components to endothelial cells plasmalemma was monitored by measurements of membrane fluidity. Namely, the fluorescent probe trimethylamonio-diphenyl hexatriene was inserted in the cell membrane and fluorescence anisotropy was determined; a higher fluorescence anisotropy means a higher rigidity of the plasmalemma. The results show that the rigidity of the endothelial cell plasmalemma increased progressively with the time of incubation (+11% to +19.5% after 24 h and 72 h, respectively for the concentration of 200 micrograms. LDL-cholesterol/dish) and with the greater amount of cholesterol in LDL (+10.9%) for 200 micrograms LDL-cholesterol/dish to +15% for 800 micrograms LDL-cholesterol/dish after 24 h incubation). In order to see if the LDL material transfer proceeded by receptor-mediated endocytosis of LDL and/or directly through aqueous solution a lysosomal inhibitor, chloroquine, was used at the concentration of 20 microM for preventing the lysosomal hydrolase activity. In the presence of this inhibitor the fluorescence anisotropy in treated endothelial cells increased by a lesser amount, suggesting an approx. 30% participation of intracellular route. Therefore, the transfer of material (probably cholesterol) from LDL to endothelial plasmalemma could take place both by receptor-mediated endocytosis and directly through the aqueous solution.

Experimental model for the quantitative estimation of transendothelial transport in vitro; a two-compartment system.

A two-compartment system was set up for the measurement of macromolecules, particles and water transfer through the monolayers of cultured endothelial cells. Experimental parameters like differential hydrostatic and osmotic pressures (delta P and delta pi, respectively) concentration gradients, temperature could be controlled. The experiments were carried out, on a cellular line of bovine aortic endothelium produced in our laboratory. Morphological and immunological investigations attested the differentiation of these cells grown on a porous support membrane separating the two compartments. The system allowed morphologic check up on the continuity of endothelial monolayer and ultrastructural studies. Hydraulic conductivity determined for endothelial monolayers was 6.86 +/- 0.85 cm. sec-1 X cm H2O-1 X 10(-7) at delta P = 10 mm Hg and 8.23 +/- 0.80 cm. sec-1 X cm H2O-1 X 10(-7) at delta P = 30 mm Hg, which proves an adequate functional integrity. Investigations using successive radioactive pulses on the same culture were possible without notable residual radioactivity between pulses.

Evidence that some membrane ligands modulate the plasmalemma fluidity of endothelial cells in culture.

The effect of some membrane ligands on the plasmalemmal fluidity of endothelial cells from bovine aorta in culture was investigated. The ligands used were: cationic ferritin (pI 8.5), soybean agglutinin, concanavalin A, wheat germ agglutinin, as well as glutaraldehyde at different concentrations. The fluidity probe employed was 1,6-diphenyl-1, 3, 5-hexatriene (DPH) and the parameter determined to quantify the fluidity was fluorescence steady-state anisotropy. The optimum time interval required by the insertion of the fluorescent probe in plasmalemma and the appropriate density of cells in the sample were determined. Rigidisation of plasmalemma was detected following its interaction with glutaraldehyde at concentrations ranging from 0.5% to 2% (+8% to +14% relative to the controls). After exposing endothelial cells to wheat germ agglutinin, concanavalin A and cationic ferritin pI 8.5, no modifications in the steady-state DPH fluorescence anisotropy were noticed. However, plasmalemmal rigidisation of +10% to +14% relative to the controls was obtained when endothelial cells were treated with 1 mg/ml and 2 mg/ml of soybean agglutinin, respectively. The possible mechanism of membrane fluidity modulation by membrane ligands and the usefulness of such investigations are discussed.