A micropipette technique was adopted to investigate the effect of blockade of integrin betal on adhesion of hepatocellular carcinoma (HCC) cells onto type IV collagen (Col IV) coated surfaces and pseudopod protrusion of HCC cells in response to Col IV stimulation. Adhesion strength was expressed as an adhesion force, which was defined as the product of the cross sectional area and critical negative pressure needed to detach single cell away from the substrate. Chemotactic pseudopod protrusion of an HCC cell was evaluated using a dual-pipette set-up, in which two pipettes filled with Col IV solution were positional in close contact with the same cell and pseudopod protrusion into each pipette was viewed dynamically and recorded with a tape recorder. The lengths of pseudopods were measured and plotted against time to obtain a pseudopod growth curve. The integrin beta1 subunit on the surfaces of HCC cells was analyzed by flow cytometry. The results showed that the adhesion forces for HCC cells adhering on 5 microg/ml Col IV coated surfaces were 932 +/- 134 (x 10(-10) N, n = 60). Upon treatment of HCC cells with Anti-CD29 in a protein concentration of 5 microg/ml and 10 microg/ml, the value decreased significantly to 449 +/- 119 (x 10(-10) N, n = 60) and 220 +/- 78 (x 10(-10) N, n = 55), respectively. In dual pipette chemotaxis experiment, when the two pipettes were filled with Col IV in an identical concentration of 600 microg/ml, pseudopods extended from the HCC cell into each of the pipettes nearly symmetrically, i.e., with nearly identical maximum pseudopod length and similar pseudopod growth curves. Upon addition of Anti-CD29 to one of the pipettes in a protein concentration of 20 microg/ml, pseudopod protrusion was blocked nearly completely while protrusion into the opposite pipette became more evidently, with larger maximum length. Expression of integrin beta1 was up to 95.78% to cells chosen in the experiment. These results suggested that integrin beta1 subunit was important constituent receptor subunit for mediating HCC cell adhesion and chemotactic pseudopod protrusion to Col IV.
Antibodies, Monoclonal ; pharmacology ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Adhesion ; Chemotaxis ; immunology ; Collagen Type IV ; metabolism ; Humans ; Integrin beta1 ; biosynthesis ; immunology ; Liver Neoplasms ; metabolism ; pathology ; Neoplasm Invasiveness ; Receptors, Very Late Antigen ; biosynthesis ; immunology ; Tumor Cells, Cultured

Anodic spark deposition method(ASD) surface treated titanium implant possesses a considerable osteoconductive potential that promoting a high level of implant osseointegration in normal bone. The purpose of this study was to observe the ASD implant's osseointegration in the osteoporosis-induced animal model. Twenty four rats, 10 weeks of age, were ovarectomized and 5 weeks later divided into two groups : ASD implant group and control implant group. Titanium screw implants (diameter; 2.0 mm, length, 3.5 mm; pitch-height, 0.4 mm) were designed for this study. Experimental implants were ASD treated and no treatment on control implants. ASD implants and control implants were placed in to left tibiae of rats. The rats were sacrificed at different time interval(1, 2, 4 and 8 weeks after implantation) for histopathologic observation and immunohisto -chemistrical observation, with collagen type I, fibronectin, integrin alpha2beta1 and integrin alpha5beta1 antibodies. The results obtained from this study were as follow: 1. Histopathologic findings, overall tissue response and the pattern of bone formation in both groups were similar. In ASD group, more newly formed bone was seen at 1 week and 2weeks than control group. 2. The levels of type I collagen and fibronectin expression were the most abundant at 2weeks and decreased gradually in both groups. Fibronectin and type I collagen expression in ASD group were stronger than control group but no significance. 3. The levels of integrin alpha2beta1 and Integrin alpha5beta1 expression were most abundant at 2 weeks and decreased gradually in both groups. No significant difference was observed in both groups. From this results, anodic oxidized titanium implants were more advantages in early stage of bone formation than control group, but have no significance in tissue responses and late bone formations. It could be stated that although anodic oxidized titanium implant possesses considerable osteoconductive potential but in osteoporotic bone condition dental implant procedure should performed after improving or treating the osteoporotic bone condition.
Animals ; Antibodies ; Collagen Type I ; Dental Implants ; Fibronectins ; Implants, Experimental ; Integrin alpha2beta1 ; Integrin alpha5beta1 ; Models, Animal ; Osseointegration ; Osteogenesis ; Osteoporosis ; Rats ; Tibia ; Titanium

OBJECTIVE: The adhesion molecule that mediate cell-cell and cell-extracellular matrix adhes.ion provides very important role in growth and differentiation of cells and tissue. VLA integrin is a prototype of adhesion molecule which participate in cell-cell and cell-extracellular matrix interacton, especially for collagen, laminin and fibronectin. The biologic functions of VLA-integrin are very diverse. Cartilage is the target tissue of various arthritides including rheumatoid arthritis and osteoarthritis and the process of homeostasis in cartilage matrix may be very important in preservation of cartilage. Although VLA integrin may participate in the process of cartilage degeneration and repair mechanism, tissue.distribution and exact role of VLA integtin in cartilage was not yet clearly defined. METHODS:Immunohistochemical analysis of VLA-integrin in cryostat section of articular cartilage was conducted using monoclonal antibody and avidin-biotin complex method. Analysis was performed in 10 rheumatoid arthritis specimens, 7 osteoarthritis specimens and 1 normal control. RESULTS: 1) Normal cartilage showed strong and diffuse stain with CD29, CD51 and moderate stain of VLA-5. Staining pattern of VLA-1 and 3 was inconstant and weak in intensity. 2) The intensity of VLA expression in articular cartilage of osteoarthritis was upregulated chiefly in CD29, CD51 and slightly in VLA-5. The positive rate of VLA-1 and 3 was similar to that of normal cartilage though the intensity was increased especially at cluster of chondrocytes. 3) VLA-integrin expression of rheumatoid arthritis cartilage was similar to that of osteoarthritis cartilage. CONCLUSION: VLA integrins functioning as fibronectin receptor such as VLA-5 and alpha, beta1 were upregulated in osteoarthritis and rheumatoid arthritis. Intensity was increased at clusters of chondrocytes. It was able to presume from above findings that VLA molecule has some role in the maintenance and repair of articular cartilage. The regulation of expression by cytokines and growth factors and exact function of VLA molecule in cartilage have to be elucidated.
Arthritis ; Arthritis, Rheumatoid ; Cartilage ; Cartilage, Articular* ; Chondrocytes ; Collagen ; Cytokines ; Fibronectins ; Homeostasis ; Integrin alpha1beta1 ; Integrin alpha5beta1 ; Integrins ; Intercellular Signaling Peptides and Proteins ; Laminin ; Osteoarthritis

To investigate the effect of vascular endothelial growth factor (VEGF) on beta1 integrin (VLA-4 and VLA-5) activation ability in K562 cells transfected with antisense VEGF121 cDNA, K562 cells were transfected with antisense (As), sense (S) and vector (V, pcDNA(3)). Flow cytometry was used to evaluate the expression rate of VLA-4 (CD49d/CD29) and VLA-5 (CD49e/CD29) and beta1 integrin activation ability in the transfected K562 cells. The results showed that the expression rates of VLA-4 and VLA-5 were more than 92% in the transfected K562 cells and there was no difference among the K562/V, K562/S and K562/As cells. However, beta1 integrin activated 9EG7 expression rate in K562/As cells was higher than that in K562/V cells [(75.6 +/- 10.5)% vs (41.2 +/- 2.1)%, P < 0.01)] after activation with beta1 integrin activator 8A2. It is concluded that function of beta1 integrin to be activated is restored in K562 cells transfected with antisense VEGF121 cDNA.
DNA ; genetics ; DNA, Antisense ; genetics ; Endothelial Growth Factors ; genetics ; metabolism ; Flow Cytometry ; Humans ; Integrin alpha4beta1 ; biosynthesis ; Integrin alpha5beta1 ; biosynthesis ; Intercellular Signaling Peptides and Proteins ; genetics ; metabolism ; K562 Cells ; Lymphokines ; genetics ; metabolism ; Transfection ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

We assessed the cytokine combinations that are best for ex vivo expansion of cord blood (CB) and the increment for cell numbers of nucleated cells, as well as stem cells expressing homing receptors, by an ex vivo expansion of cryopreserved and unselected CB. Frozen leukocyte concentrates (LC) from CB were thawed and cultured at a concentration of 1x10(5)/mL in media supplemented with a combination of SCF (20 ng/mL)+TPO (50 ng/mL)+FL (50 ng/mL)+/-IL-6 (20 ng/mL)+/-G-CSF (20 ng/mL). After culturing for 14 days, the expansion folds of cell numbers were as follows: TNC 22.3+/-7.8~26.3+/-4.9, CFU-GM 4.7+/-5.1~11.7+/-2.6, CD34+CD38- cell 214.0+/-251.9~464.1+/-566.1, CD34+CXCR4+ cell 4384.5+/-1664.7~7087.2+/-4669.3, CD34+VLA4+ cell 1444.3+/-1264.0~2074.9+/-1537.0, CD34+VLA5+ cell 86.2+/-50.9~ 113.2+/-57.1. These results revealed that the number of stem cells expressing homing receptors could be increased by an ex vivo expansion of cryopreserved and unselected CB using 3 cytokines (SCF, TPO, FL) only. Further in vivo studies regarding the engraftment after expansion of the nucleated cells, as well as the stem cells expressing homing receptors will be required.
ADP-ribosyl Cyclase/metabolism ; Antigens, CD/metabolism ; Antigens, CD34/metabolism ; Cryopreservation ; Fetal Blood/*cytology ; Humans ; Integrin alpha4beta1/metabolism ; Integrin alpha5beta1/metabolism ; Membrane Proteins ; Receptors, CXCR4/metabolism ; Receptors, Lymphocyte Homing/*metabolism ; Research Support, Non-U.S. Gov't ; Stem Cell Factor ; Stem Cells/*cytology/*metabolism ; Thrombopoietin

Adhesion to extracellular matrix plays important roles in the regulation of survival, proliferation, differentiation and homing of hematopoietic cells and is regulated by a wide variety of growth factors, adhesion receptors and other ligands that mediate the cell to matrix and cell to cell interaction. Stem cell factor (SCF) plays important roles in the regulating growth and self-renewal of hematopoietic stem/progenitor cells. In the report, the effects of stem cell factor on the adhesion of hematopoietic cells to fibronectin were observed by using a hematopoietic growth factor dependent cell line-Mo7e. Results showed that Mo7e cells express the very late antigen VLA-4 (beta1 alpha4) and VLA-5 (beta1 alpha5) integrins. The expression of the SCF receptor (c-kit) was also detected in the Mo7e cells. SCF enhances the adhesion of Mo7e cells to fibronectin in a concentration dependent manner. SCF enhanced adhesion of Mo7e cells to fibronectin was blocked by anti-beta1, alpha4 and alpha5 antibodies. Addition of PI-3 kinase inhibitors also blocked the adhesion of Mo7e cells to fibronectin induced by SCF. It was concluded that SCF enhances the adhesion of Mo7e cells to fibronectin, and this process is mediated by integrins and PI-3 kinase pathway.
Antibodies, Monoclonal ; pharmacology ; Cell Adhesion ; drug effects ; Chromones ; pharmacology ; Dose-Response Relationship, Drug ; Enzyme Inhibitors ; pharmacology ; Fibronectins ; metabolism ; Hematopoietic Stem Cells ; drug effects ; metabolism ; Humans ; Integrin alpha4beta1 ; immunology ; metabolism ; Integrin alpha5beta1 ; immunology ; metabolism ; Morpholines ; pharmacology ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors ; metabolism ; Stem Cell Factor ; pharmacology ; Tumor Cells, Cultured

BACKGROUND AND OBJECTIVES: Adenosine diphosphate (ADP), which is usually secreted from activated platelets, may activate integrins on vascular smooth muscle cells, resulting in adhesion and proliferation. Integrins, mediating the ADP-stimulated adhesion and proliferation of vascular smooth muscle cells, was investigated in this study. MATERIALS AND METHODS: Prothrombin (PT) and bone sialoprotein (BSP) were used as activation-dependent ligands in an adhesion assay. The adhesion of human aortic smooth muscle cells (HASMC) were measured after ADP stimulation, using ligand-coated 24-well plates. The 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the ADP-stimulated proliferation of the HASMC.RESULTS: ADP activated the HASMC to increase their adhesion to the PT or BSP, and their proliferation in a dose-dependent manner. The adhesion of the ADP-stimulated HASMC to the PT was completely blocked by P5H9, a blocking monoclonal Ab (mAb) to integrin alphavbeta5 (92% inhibition), but was only slightly inhibited by LM609, a blocking mAb to integrin alphavbeta3 (30% inhibition). The adhesion of the ADP-stimulated HASMC to the BSP was partially inhibited by both P5H9 (46% inhibition) and JBS5, a blocking mAb to integrin alpha5beta1 (75% inhibition), but was not affected by c7E3, a blocking mAb to integrin beta3. The ADP-stimulated proliferation of the HASMC was inhibited by both c7E3 and LM609 (98% and 93% inhibition, respectively), but not by either P1F5, a blocking mAb to integrin alphavbeta5 or JBS5. CONCLUSION: These results indicate the different roles of integrins on vascular smooth muscle cells after ADP stimulation; the integrins alphavbeta5 and alpha5beta1 for adhesion, and the integrin alphavbeta3 for proliferation.
Adenosine Diphosphate ; Humans ; Integrin alpha5beta1 ; Integrin alphaVbeta3 ; Integrin beta3 ; Integrin-Binding Sialoprotein ; Integrins* ; Ligands ; Muscle, Smooth, Vascular* ; Myocytes, Smooth Muscle ; Negotiating* ; Prothrombin

Drug-induced gingival overgrowth (DIGO) is characterized by fibrous gingival hyperplasia and increased gingival volume. DIGO is histologically associated with proliferation of cells and deposition of extracellular matrices, particularly collagen. Integrin α2β1 is related to collagen phagocytosis and involved in the occurrence and progression of DIGO. This paper reviews the progress of research on the relationship between integrin α2β1 and DIGO.
Collagen ; Gingiva ; Gingival Overgrowth ; Humans ; Integrin alpha2beta1 ; Phagocytosis

OBJECTIVETo study the expression of alpha5beta1 integrin in the abnormal scars and its role and significance in the formation and development of abnormal scars.

METHODSThe expression of alpha5beta1 integrin was observed in hypertrophic scar (15 samples), keloid (15 samples) and normal skin (10 samples) with SP immunohistochemical method and colloidal gold immuno-electron microscopic technique. The data were semi-quantitatively analyzed.

RESULTSThe expression levels of alpha5beta1 integrin in the fibroblasts of keloids and hypertrophic scars were higher than normal skin; the expression of alpha5beta1 integrin in the fibroblasts of keloids was higher than hypertrophic scars (P < 0.01).

CONCLUSIONThe alpha5beta1 integrin appears to have close relation to the formation and development of abnormal scars. To find a way to decrease the expression level of alpha5beta1 integrin in fibroblasts may be a new approach to inhibit scar hypertrophy.

Cicatrix ; metabolism ; pathology ; Humans ; Immunohistochemistry ; Integrin alpha5beta1 ; analysis ; metabolism ; Keloid ; metabolism ; pathology ; Microscopy, Immunoelectron ; Skin ; chemistry ; pathology ; ultrastructure

OBJECTIVETo explore the correlation between the expression of integrin subunits alpha5 and beta1 in prostate cancer (PCa) and its clinicopathological data including tumor grade and clinical stage.

METHODSExpressions of integrin subunits alpha5 and beta1 were examined in 30 cases of PCa and 30 cases of normal prostatic tissues by immunohistochemical assay.

RESULTSExpressions of integrin subunits alpha5 and beta1 in PCa were lower than those in normal prostatic tissues (P <0.05) respectively.

CONCLUSIONCompared with normal prostatic tissues, expressions of integrin subunits alpha5 and beta1 in PCa were rather weaker or even faded. Expressions of integrin subunits alpha5 and beta1 revealed an positive correlation with tumor's Gleason grade and negative with clinical TNM stage. The results indicate that integrin subunits alpha5 and beta1 have potential values in the diagnosis and are predictable indices in the proliferation of PCa.

Aged ; Aged, 80 and over ; Animals ; Humans ; Integrin alpha5 ; biosynthesis ; Integrin alpha5beta1 ; biosynthesis ; Male ; Mice ; Middle Aged ; Neoplasm Staging ; Prostatic Hyperplasia ; metabolism ; Prostatic Neoplasms ; metabolism ; pathology