VCAM-1 expression on bone marrow stromal cells from patients with myelodysplastic syndromes.

We studied the expression of VCAM-1 adhesion molecules on stromal cells from the bone marrow of patients with myelodysplastic syndromes, healthy donors, and patients with chronic myeloproliferative diseases and acute leukemias. Expression of adhesion molecule on mesenchymal stromal cells from the bone marrow of patients and healthy donors was evaluated after 2-4 passages by the methods of immunoprecipitation and electrophoresis. VCAM-1 expression in the majority of patients with myelodysplastic syndromes was lower than in healthy donors. At the same time, VCAM-1 expression was not identified on mesenchymal cells from acute leukemia patients. VCAM-1 expression on cells from patients with chronic myeloproliferative diseases did not differ from that in healthy donors. We conclude that VCAM-1 synthesis in bone marrow stromal cells is impaired in patients with myelodysplastic syndromes and acute leukemias. These changes can be followed by the loss of relationships between hemopoietic cells and stromal microenvironment in bone marrow niches. Hemopoietic cells gain the ability for uncontrolled growth, which results in progression of the disease.

Defective adhesion and homing of hemopoietic precursors from the bone marrow of patients with myelodysplasia to stromal cells of normal bone marrow cultures.

Hemopoietic precursors from the bone marrow of patients with myelodysplastic syndrome were characterized by lower adhesion to normal stromal sublayer compared to bone marrow precursors from healthy donors, while adhesion to fibroblast monolayer and fibronectin was similar in bone marrow cells from patients and donors. In vitro experiments showed that the percentage of adherent hemopoietic precursors from the bone marrow of patients with myelodysplastic syndrome in normal stromal sublayer and fibroblasts was lower compared to healthy donors. The decrease in adhesive activity of hemopoietic precursors from the bone marrow of patients with myelodysplastic syndrome probably contributes to impairment of cell-cell interactions in the bone marrow of these patients.

[In vitro cell adhesion and integrin expression by calcium ionophore-treated mononuclear cells from patients with acute myeloid leukemia]. / Adgezivnye svoistva i ékspressiia integrinov kletkami bol'nykh ostrymi mieloidnymi leikozami, stimulivannymi v kul'ture ionoforom dlia ionov kal'tsiia.

As shown elsewhere, cultured acute myeloid leukaemia blasts acquire certain characteristics of dendritic cells upon stimulation with cytokines and calcium ionophore. The ability of leukaemia-derived dendritic-like cells to express immune costimulatory molecules and dendritic cell marker CD83 has been extensively investigated. Although migratory capacity is a major attribute of dendritic cells, the ability of in vitro modified blasts for adhesion, chemotaxis and homing remain elusive. In the present paper, we show that after stimulation with calcium ionophore acute myeloid leukaemia blasts as well as normal dendritic cell precursors demonstrate increased capacity of binding fibronectin and denatured collagen. The expression pattern of integrins on dendritic-like leukaemic cells in general closely resembles that of monocyte-derived dendritic cells, however, variation in cell properties isolated from blood of individual patients are observed.

[Limited proteolysis of integrin alpha v beta3 from human placenta]. / Ogranichennyi proteoliz integrina alpha v beta3 iz platsenty cheloveka.

Purification of alpha v beta 3 integrin from human placenta with successive usage of two affinity sorbents--immobilized monoclonal antibodies to alpha v beta 3 integrin and immobilized RGD-containing decapeptide allowed to purify this integrin's partially degraded fraction, that was nevertheless able to interact with its ligand. During the incubation of partially degraded alpha v beta 3 integrin at 37 degrees C its further degradation went on. Addition of serine proteinase inhibitors: (phenylmethilsulfonyl fluoride, leupeptin and aprotinin) completely suppressed integrin further degradation of alpha v beta 3. In preparations of intact and partially degraded alpha v beta 3 integrin specific activity of two serine proteinases--urokinase and dipeptidilpeptidase IV--was discovered. alpha v beta 3 integrin, undergoing limited proteolysis, had lesser affinity towards RGD peptide, that intact integrin. The results show, that alpha v beta 3 integrin from human placenta co-purifies with serine proteinases. It is suggested that a definite part of functionally active alpha v beta 3 integrin, extracted from human placenta by triton X-100, forms a stable complex with serine proteinases.

[Electrophoretic characteristics of integrin++ beta1 subunit from human smooth muscles]. / Electroforeticheskoe povedenie beta1 sub''edinitsy integrina iuz gladkikh myshts cheloveka.

Total fraction of beta 1 integrin family was isolated from human smooth muscle by affinity chromatography on immobilized anti-beta 1 monoclonal antibodies. SDS-PAGE analysis and subsequent immunoblotting demonstrated that integrin samples contain unknown before high molecular mass (205 kD-nonreduced and 230 kD-reduced) material immunologically related to beta 1 integrin subunit. One dimensional peptide mapping showed that the 205 kD protein is not a novel beta 1 related integrin subunit, but a beta 1 integrin subunit dimer. Reduction of electrophoretic samples with dithiothreitol led to the removal of the major part of the beta 1 immunoreactive material from 205 kD to 130 kD region, indicating a disulfide nature of B1 integrin subunit dimer. The 230 kD protein turned out to be an only partly reduced beta 1 integrin disulfide bonded dimer. Possible in vivo existence of the disulfide bonded dimer and oligomer integrin forms is discussed.

Beta 1 integrin subunit dimerization via disulfide bonds.

Integrins of the beta 1 family were isolated from human smooth muscle. SDS-PAGE analysis and subsequent immunoblotting demonstrated that integrin samples contain a protein immunologically related to beta 1 integrin subunit with the previously undescribed apparent molecular mass 205 kD. One-dimensional peptide mapping showed that the 205 kD protein is not a novel beta 1 related integrin subunit, but a beta 1 integrin subunit dimer. After reduction the major part of the beta 1 immunoreactive material migrated from the 205 kD to 130 kD region, indicating that beta 1 integrin subunit dimers were formed via disulfide bonds. When electrophoretically pure beta 1 monomer and dimer forms were analized it was found that during SDS-PAGE about 30% of beta 1 integrin subunit monomers were organized into dimers while approximately 70% of the beta 1 dimer form was partly disrupted into monomers. It was suggested that this steady-state process is a result of a reversible reaction between intra- and intermolecular disulfide bonds. Possible in vivo dimerization of integrins via disulfide bonds is discussed.

[Isolation and characteristics of ligand specificity of VLA-1 integrin from human smooth muscles]. / Vydelenie i kharakteristika ligandnoi spetsifichnosti VLA-1 integrina iz gladkikh myshts cheloveka.

Using affinity chromatography with immobilized monoclonal antibodies to the beta 1-subunit of human integrin, a total integrin fraction (subfamily beta 1) was isolated from the detergent extract of human smooth muscle (uterus). Immunoprecipitation and immunoblotting with specific antibodies revealed integrins VLA-1 and VLA-5. The former was isolated in a homogeneous state by chromatography on immobilized type I collagen in the presence of 1 mM Mn2+. The pure receptor yield was 2-4 mg per 400 g of smooth muscle tissue. Analysis of substrate specificity of VLA-1 in the liposome test revealed that this integrin possesses a broad spectrum of ligand specificity and can interact via a Ca2+, Mg(2+)-dependent mechanism with interstitial collagens of I, II and III types and with basal membrane proteins (type IV collagen and laminin). VLA-1 does not interact with fibronectin, thrombospondin or albumin. Denaturation of type I collagen decreases the liposome binding 5-7-fold. The peptide Gly-Arg-Gly-Asp-Ser-Pro added to the incubation mixture does not inhibit the liposome interaction with incorporated VLA-1 integrin, type I collagen and laminin.

Human smooth muscle VLA-1 integrin: purification, substrate specificity, localization in aorta, and expression during development.

A membrane glycoprotein complex was isolated and purified from human smooth muscle by detergent solubilization and affinity chromatography on collagen-Sepharose. The complex was identified as VLA-1 integrin and consisted of two subunits of 195 and 130 kD in SDS-PAGE. Liposomes containing the VLA-1 integrin adhered to surfaces coated with type I, II, III, and IV collagens, Clq subcomponent of the first component of the complement, and laminin. The liposomes specifically adhered to these proteins in a Ca2+, Mg2(+)-dependent manner, but did not bind to gelatin, fibronectin, and thrombospondin substrates. The expression of VLA-1 integrin in different human tissues and cell types, and during aorta smooth muscle development was studied by SDS-PAGE, and subsequent quantitative immunoblotting was performed with antibodies recognizing alpha 1 and beta 1 subunits of the VLA-1 integrin. A high level of VLA-1 integrin expression was an exceptional feature of smooth muscles. Fibroblasts, endothelial cells, keratinocytes, striated muscles, and platelets contained trace amounts of VLA-1 integrin. In the 10-wk-old human fetal aorta, VLA-1 integrin was found only in smooth muscle cells whereas mesenchymal cells, surrounding aortic smooth muscle cells, were VLA-1 integrin negative. By the 24th wk of gestation, the amount of VLA-1 integrin was significantly reduced in the aortic media (4.3-fold for alpha 1 subunit and 2.5-fold for beta 1 subunit) compared with that in the 10-wk-old aortic smooth muscle cells. After birth, the expression of VLA-1 integrin increased and in the 1.5-yr-old child aorta the VLA-1 integrin level was almost the same as in adult aortic media. Smooth muscle cells from intimal thickening of adult aorta express five times less alpha 1 subunit of VLA integrin that smooth muscle cells from adult aortic media. In primary culture of aortic smooth muscle cells, the content of the VLA-1 integrin was dramatically reduced and subcultured cells did not contain VLA-1 integrin at all.

[Fibronectin distribution during the transdifferentiation and proliferation of eye cells after retinal detachment and removal of the crystalline lens in tritons]. / Raspredelenie fibronektina v protsesse transdifferentsirovki i proliferatsii kletok glaza posle otsloiki setchatki i udaleniia khrustalika u tritonov.

Expression of fibronectin (Fn) during eye tissue regeneration in the newt after retinal detachment and lens removal was studied by immunohistochemistry. Proliferation of cells involved in eye tissue regeneration was studied using autoradiography. Fn was detected around the cell membranes of undifferentiated proliferating and migrating cells in ciliary body of the iris and growth zone of the retina. Redistribution of Fn was observed in proliferating cells of the dorsal iris participating in lens regeneration. Fn appeared on the apical surface of proliferating redifferentiating pigment epithelium (PE) cells at the periphery of the eye and over the whole surface of proliferating PE cells in the central part of the eye. The Fn level in the Bruch's membrane decreased in the area of transdifferentiating cells detachment from PE layer (in the lower part of the eye) but continued to be stable in the area of PE cell redifferentiation (at the periphery of the eye). The role of Fn is discussed in relation to transdifferentiation, proliferation and migration of cells in the regenerating eye.

[Blood plasma fibronectin does not influence fibroblast-induced collagen gel contraction]. / Fibronektin iz plazmy krovy ne vliiaet na kontraktsiiu kollagenovogo gelia, vyzyvaemuiu fibroblastami.

Human embryo fibroblasts were cultured within three-dimensional collagen gel in the Eagle medium supplemented with 10% calf serum. Addition of fibronectin (Fn) to the nutrient medium neither stimulated nor inhibited gel contraction by fibroblasts. Gel contraction also proceeded in the medium containing ultroser G or Fn exhausted calf serum. Addition of 10 or 50 micrograms/ml of Fn to the medium with ultroser G exerted no effect on the process of gel contraction. Addition of antibodies prepared against Fn (70 micrograms/ml) and tetrapeptide RGDS (100 and 500 micrograms/ml) to the medium supplemented with Fn exhausted calf serum did not change the rate of collagen gel contraction. The results obtained suggest that the rate of collagen gel contraction by fibroblasts is not dependent on the presence of exogenous fibronectin.

[Properties and conditions of formation of the fibronectin-collagen complex in a culture of human embryo fibroblasts]. / Nekotorye svoistva i usloviia formirovaniia fibronektin-kollagenovogo kompleksa v kul'ture émbrional'nykh fibroblastov cheloveka.

In our previous study the macromolecular complexes mostly consisting of fibronectin and procollagen were isolated from human fibroblast culture media using immobilized antibodies against fibronectin. At present an attempt was made to elucidate at what stage the formation of the fibronectin-collagen complex occurs--either in the course of incubation of the immobilized antibodies with labelled proteins secreted by fibroblasts, or in the extracellular space while labelling fibroblasts, or intracellularly. The results obtained show that the fibronectin-collagen complex: 1) pre-exists even before incubation with the immobilized antibodies and 2) it is of intracellular origin. Thus, the considerable amount of the fibroblast-secreted fibronectin (no less than 20%) is released from the cell not in the free form but in the complex with procollagen. It was suggested that the fibronectin-collagen complex presents a stage in the formation of the insoluble extracellular matrix.

[Immunohistochemical study of fibronectin localization during the formation of the vascular coat under the influence of the pigment epithelium in chick embryos]. / Immunogistokhimicheskoe issledovanie lokalizatsii fibronektina pri formirovanii sosudistoi obolochki pod vliianiem pigmentnogo épiteliia u kurinykh zarodyshei.

The role of fibronectin (FN) in cell interactions of retinal pigment epithelium (RPE) and mesenchyme surrounding the optic cup during choroid formation in chick embryos was studied by indirect immunofluorescence using antibodies against FN. Experimental coloboma of retina and choroid was used as a model. During the initial stages of coloboma the regions structured like retina rudiment appear in the outer layer of the optic cup. Such regions were formed in microphthalmic eyes obtained by excision of lens from the eyes of 3.5 day old chick embryos (stage 21). At stage 21 bright FN-specific immunofluorescence was observed in basal membrane located along the external surface of the normally differentiated RPE. Later on, FN-specific immunofluorescence appeared in mesenchyme condensing along the RPE. The most intensive FN-specific immunofluorescence was observed in chorio-capillary layer of choroid after 5-7 days of incubation. In microphthalmic eyes retina-like regions of RPE and adjacent mesenchyme showed negative reaction, and the choroid was not formed from the adjacent mesenchyme in such zones. The data obtained suggest that the presence of normally differentiated RPE producing FN-containing basal membrane is necessary for the formation of chorio-capillary layer of the choroid in chick embryos.

[The use of immobilized antibodies against fibronectin for isolation of macromolecular complexes of fibronectin with other secreted proteins from human fibroblasts]. / Ispol'zovanie immobilizovannykh antitel k fibronektinu dlia vydeleniia makromolekuliarnykh kompleksov fibronektina s drugimi sekretiruemymi belkami fibroblastov cheloveka.

Interaction of labelled proteins from human fibroblast culture and the antibodies to fibronectin, which were used as soluble and Sepharose-immobilized preparations, was studied. Electrophoretic mobilities of the proteins bound to antibodies were compared. Soluble antibodies bound only fibronectin, while immobilized antibodies bound, except for fibronectin, other low molecular proteins. After treatment of the cultural proteins with bacterial collagenase two main components, besides fibronectin, were eliminated, thus suggesting their collagen-like nature. Two dimers of fibronectin per one trimer of procollagen were present in the material bound to immobilized antibodies to fibronectin. Control studies demonstrated that the data obtained were not an artefact occurring due to sorption of proteins on Sepharose or to unspecific actions of antibodies.

Collagen and fibronectin synthesis by trisomic and triploid fibroblasts from human spontaneous abortuses.

Collagen and fibronectin synthesis by trisomic and triploid fibroblasts derived from human spontaneous abortuses was studied. It was demonstrated that the level of fibronectin and collagen production in fibroblasts with trisomy 7, trisomy 9, and triploidy was reduced as compared with diploid cells. A correlation between this observation and an increased rate of intracellular 14C-procollagen degradation was also established for the anomalous strains. No difference in hydroxylation of 14C-proline residues in alpha 1(I) and alpha 2(I) collagen chains and no fluctuation in the collagen type (I): type III ratio was found in the strains with the abnormal karyotypes. It was concluded that differentiation of the abnormal fibroblasts was impaired. The data also favour the hypothesis that the deficiency of the fibroblasts in producing proteins may account for a variety of anatomic abnormalities of embryos.

[Biosynthesis of fibronectin by human embryonal fibroblasts transformed by SV-40 virus]. / Biosintez fibronektina chelovecheskimi émbrional'nymi fibroblastami, transformirovannymi virusom SV-40.

Fibronectin biosynthesis by human embryonic fibroblasts transformed with virus SV-40 was studied in intact cells and in a cell-free protein synthesizing system on free and membrane-bound polyribosomes isolated from these cells. It was found that fibronectin release from transformed fibroblasts into the culturing medium was decreased 4.5-fold, while its per cent content--2-fold. The amount of fibronectin precipitated by antibodies in the course of an immunoprecipitation reaction in transformed cells appeared to be somewhat higher than in normal cells, although when expressed on a per cent basis this content was decreased only 1.5-fold. However, the content of fibronectin monomer with Mr = 220 kD exceeded that in normal fibroblast cell material 1.6 times. Study on fibronectin biosynthesis in a cell-free system revealed that in transformed cells 45% of fibronectin is synthesized on free polyribosomes as compared to 13% in normal fibroblasts. It is assumed that the decreased fibronectin biosynthesis in human fibroblasts transformed with virus SV-40 results in spatial uncoupling of polyribosomes and membrane structures responsible for protein transport from the cell, as a result of which a significant part of fibronectin synthesized by transformed fibroblasts undergoes intracellular degradation.

[Fibronectin-synthesizing activity of free and membrane-bound polyribosomes from human fibroblasts and chick embryos]. / Fibronektinsinteziruiushchaia aktivnost' svobodnykh i membransviazannykh poliribosom iz chelovecheskikh fibroblastov i kurinykh émbrionov.

Using immunochemical methods, the fibronectin-synthesizing activity of membrane-bound and free polyribosomes in a cell-free system was studied. It was demonstrated that fibronectin biosynthesis on membrane-bound polyribosomes from human embryonic fibroblasts makes up to 4.9%, while that from chicken embryos--1.1% of the total amount of the de novo synthesized proteins (as compared to 1.0 and 0.3% in free polyribosomes, respectively). Fibronectin monomers (Mr = 330 kD) were detected only in the newly synthesized (in the presence of spermidine) material of the cell-free system containing heavy fractions of membrane-bound polyribosomes.

[Changes in fibronectin biosynthesis in human embryonal fibroblasts with trisomy for chromosomes 7 and 9]. / Izmenenie biosinteza fibronektina v émbrional'nykh chelovecheskikh fibroblastakh s trisomiei po khromosomam 7 i 9.

Biosynthesis of fibronectin in 2 strains of aneuploid human fibroblasts with trisomy by chromosomes 7 and 9 was about 2-fold decreased as compared with normal diploid fibroblasts. The most distinct decrease of the fibronectin biosynthesis was observed at the logarithmic phase of the cell culture growth. The cells with anomalous karyotype exhibited also the decreased rate of secretion of the impulse labelled fibronectin. The decrease in the rate of fibronectin and collagen production (main proteins of intercellular substance) in fibroblasts with the chromosomes anomalies might be responsible for irreversible impairments of the embryo development, resulting in spontaneous abortion.

[Intracellular localization of fibronectin-synthesizing polyribosomes using I125 collagen]. / Izuchenie vnutrikletochnoi lokalizatsii fibronektinsinteziruiushchikh poliribosom pri pomoshchi [125I]kollagena.

Interaction of 125I-collagen with fibronectin-synthesizing polyribosomes, isolated from 9 days old chicken embryos, was studied. The interaction was maximal at the ratio of 0.1 microgram 125I-collagen and 5 ou of polyribosomes. Specific reaction of 125I-collagen with fibronectin polypeptides growing on polyribosomes was estimated by means of antibodies to fibronectin inhibiting the complex formation of polyribosomes-125I-collagen. The antibodies were shown to decrease the reaction rate of total polyribosomes (free and membrane-bound) with 125I-collagen by 25.4% and of free polyribosomes - by 6.7%. The data obtained suggest that fibronectin is synthesized mainly on polyribosomes, bound with the membranes of endoplasmic reticulum.

[Reaction between 125I-collagen and growing fibronectin polypeptides of chick embryo polyribosomes]. / Vzaimodeistvie 125I-kollagena s rastushchimi polipeptidami fibronektina polirobosm kurinykh émbrionov.

Polyribosomes from chicken embryos and 125I-collagen were shown to form complexes, which might be isolated from free 125I-collagen by means of centrifugation. The fraction of rapidly sedimenting polyribosomes was the most active in formation of complexes with 125I-collagen. The complexes ribosomes-125I-collagen appeared after destruction of polyribosomes-125I-collagen complexes by means of RNAase. Treatment of the complexes with puromycine led to liberation of about 40% of 125I-collagen. Antibodies against fibronectin inhibited the complex formation by about 30%. The data obtained suggest that the growing fibronectin polypeptides participate in formation of at least 30% of the complexes.