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PubMed1998

Differential expression and distribution of focal adhesion and cell adhesion molecules in rat hepatocyte differentiation.

Hepatocytes in primary culture enter into clonal proliferation in the chemically defined hepatocyte growth medium in the presence of hepatocyte growth factor and epidermal growth factor. Hepatocyte proliferation is associated with loss of differentiated gene expression. Overlay of matrix derived from Engelbreth-Holm-Swarm mouse sarcoma (Matrigel) on proliferating hepatocytes induces reexpression of the hepatic differentiation marker genes. To explore the role of matrix in the differentiation process of hepatocytes, we examined the mRNAs of fibronectin, vitronectin, and entactin in proliferating hepatocytes and Matrigel-treated hepatocytes. Fibronectin mRNA increased in proliferating hepatocytes at days 2-10 and then decreased; however, vitronectin mRNA disappeared in proliferating hepatocytes and was reexpressed in Matrigel-treated hepatocytes. We also found that focal adhesion kinase and paxillin were strongly increased in Matrigel-treated hepatocytes, and E-cadherin and beta-catenin slightly increased in Matrigel-treated hepatocytes, suggesting that both cell-to-extracellular matrix and cell-to-cell interactions may be an essential part of hepatocyte differentiation. To evaluate the distribution of focal adhesion associated molecules and cell-to-cell adhesion molecules, Triton X-100 soluble and insoluble fractions were examined at days 8, 9, 10, and 11 in proliferating hepatocytes and Matrigel-treated cells. We found that E-cadherin in Triton X-100 insoluble fractions dramatically decreased in Matrigel-treated hepatocytes; however, beta-catenin strongly increased in Triton X-100 soluble fractions of Matrigel-treated hepatocytes. These results suggest that the distribution of both focal adhesion associated molecules and cell adhesion molecules are reorganized during the process of differentiation induced by overlay of Matrigel.

Animals↗
PubMed1999

Engineered smooth muscle tissues: regulating cell phenotype with the scaffold.

Culturing cells on three-dimensional, biodegradable scaffolds may create tissues suitable either for reconstructive surgery applications or as novel in vitro model systems. In this study, we have tested the hypothesis that the phenotype of smooth muscle cells (SMCs) in three-dimensional, engineered tissues is regulated by the chemistry of the scaffold material. Specifically, we have directly compared cell growth and patterns of extracellular matrix (ECM) (e.g. , elastin and collagen) gene expression on two types of synthetic polymer scaffolds and type I collagen scaffolds. The growth rates of SMCs on the synthetic polymer scaffolds were significantly higher than on type I collagen sponges. The rate of elastin production by SMCs on polyglycolic acid (PGA) scaffolds was 3.5 +/- 1.1-fold higher than that on type I collagen sponges on Day 11 of culture. In contrast, the collagen production rate on type I collagen sponges was 3.3 +/- 1.1-fold higher than that on PGA scaffolds. This scaffold-dependent switching between elastin and collagen gene expression was confirmed by Northern blot analysis. The finding that the scaffold chemistry regulates the phenotype of SMCs independent of the scaffold physical form was confirmed by culturing SMCs on two-dimensional films of the scaffold materials. It is likely that cells adhere to these scaffolds via different ligands, as the major protein adsorbed from the serum onto synthetic polymers was vitronectin, whereas fibronectin and vitronectin were present at high density on type I collagen sponges. In summary, this study demonstrates that three-dimensional smooth muscle-like tissues can be created by culturing SMCs on three-dimensional scaffolds, and that the phenotype of the SMCs is strongly regulated by the scaffold chemistry. These engineered tissues provide novel, three-dimensional models to study cellular interaction with ECM in vitro.

Animals↗
PubMed1999

Glycoprotein Ibalpha can mediate endothelial cell migration on von Willebrand factor-containing substrata.

We have previously shown that, in addition to the vitronectin receptor (VNR, alpha(v)beta(3)), the GP Ib complex can participate in endothelial cell (EC) attachment to von Willebrand Factor (vWF) (D. A. Beacham, M. S. Cruz, and R. I. Handin, 1995, Thromb. Haemostas. 73, 309-317; D. A. Beacham, L.-P. Tran, and S. S. Shapiro, 1997, Blood 89, 4071-4077). In this study we have investigated the functional roles of these vWF receptors in the migration of untreated and TNFalpha-treated EC on vWF, a mixture of vWF and type I collagen, and on vitronectin (VN). In agreement with previous studies (D. I. Leavesley, M. A. Schwartz, M. Rosenfeld, and D. A. Cheresh, 1993, J. Cell Biol. 121, 163-170), the migration of untreated and TNFalpha-treated EC on VN was dependent entirely on the VNR. Migration of untreated EC on vWF was inhibited 10-15% by recombinant vWF-A1, the GP Ibalpha-binding domain on vWF which abrogates the platelet GP Ibalpha-vWF interaction. In contrast, migration of TNFalpha-treated EC on vWF was inhibited 50-60% by vWF-A1 or the anti-GP Ibalpha mAb AS-7 but only 20% by the anti-VNR mAb LM609. On a mixed vWF-collagen substratum, vWF-A1 inhibited untreated EC migration by 45%, and TNFalpha-treated EC migration by 75%. The possible role of EC proliferation was eliminated, since hydroxyurea completely inhibited EC proliferation without reducing migration significantly. The anti-GP Ibalpha mAb Ib1 inhibited EC migration by 50%, but reduced proliferation by only 15%. Taken together, our data demonstrate that EC migration on vWF-containing substrata involves the GP Ib complex as well as the VNR and raises the possibility that the VNR and GP Ib act cooperatively in supporting EC migration.

Antibodies, Monoclonal↗
PubMed1997

Synergistic effect of substance P with epidermal growth factor on epithelial migration in rabbit cornea.

In order to investigate the role of neural regulation in corneal epithelial healing, we examined the effect of substance P (SP) on corneal epithelial migration using an organ culture system of rabbit corneas. We investigated the synergistic effects of SP with (1) growth factors: epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and transforming growth factor-beta(TGF-beta); (2) extracellular matrix proteins: fibronectin, vitronectin, laminin, and collagen type IV; and (3) cytokines: interleukin-1alpha (IL-1alpha), IL-1beta, and interleukin-6 (IL-6). Rabbit corneal blocks were cultured in the absence or presence of various reagents for 24 hr. The corneal blocks were then fixed, dehydrated, embedded in paraffin and stained by hematoxylin-eosin, and the length of the path of epithelial migration was measured. The addition of SP alone, at concentrations up to 50 microg ml-1, did not affect epithelial migration. EGF, fibronectin, vitronectin, collagen type IV, and IL-6 stimulated epithelial migration, but bFGF, TGF-beta, laminin, IL-1alpha, and IL-1betadid not. The stimulatory effect of EGF on the epithelial migration was enhanced by the presence of SP. This synergistic effect of SP and EGF on corneal epithelial migration was abolished by the addition of an SP antagonist or enkephalinase. Other neurotransmitters (vasoactive intestinal peptide, calcitonin gene-related peptide, acetylcholine chloride, norepinephrine, serotonin) and tachykinins (neurokinin A, neurokinin B, kassinin, eledoisin, physalaemin) were examined, but none exhibited a synergistic effect with EGF. Interestingly, EGF alone stimulated the incorporation of 3H-thymidine into corneal epithelial cells, but the addition of SP with EGF did not enhance this effect. These results demonstrate that SP enhanced the EGF stimulation of corneal epithelial migration in vitro in a specific manner, suggesting a possible role of SP as a modulator of epithelial wound healing.

Animals↗
PubMed2000

Induction of posterior vitreous detachment in rabbits by intravitreal injection of tissue plasminogen activator following cryopexy.

The purpose of this study was to generate intravitreal plasmin after intravitreal injection of tissue plasminogen activator (TPA) and cryopexy, and to assess its proteolytic effect on the vitreoretinal border region.Twenty-four hr after a mild cryopexy, 25 microg recombinant tissue plasminogen activator (TPA) was injected into the vitreous cavity, the fellow eye received an intravitreal injection of the same volume of buffered salt solution. Light, scanning and transmission electron microscopy was performed in 24 eyes that underwent vitrectomy 1 week later. Plasmin was measured prior and 2 hr after intravitreal TPA injection (4 eyes). Hyaluronic acid (8 eyes) and vitronectin (4 eyes) were measured 1 week after TPA- or BSS-injection and compared to untreated controls. In all eyes treated with TPA, histopathologic examination by scanning and transmission electron microscopy demonstrated a complete detachment of the vitreous from the surface of the retina as well as from the posterior surface of the lens. After BSS-injection, vitreous cortex attachment to the retina was demonstrated in all eyes. Two hr after TPA-injection, plasmin increased to 9.75 mU ml(-1)(s.d.+/-2.3). Neither a decrease of hyaluronic acid nor an increase of transglutaminase, that might alter the vitreous structure leading to a collapse of the vitreous, were detected in treated eyes. There was no increase of vitronectin indicating proliferative activity.A temporary breakdown of the blood-retinal barrier by cryopexy combined with intravitreal injection of TPA is a sufficient technique to induce a posterior vitreous detachment enzymatically. The method may be useful prior to mechanical vitrectomy.

Animals↗
PubMed1999

Effect of all-trans-retinoic acid on integrin receptors of human cervical cancer (SiHa) cells.

Cell surface receptors have been the subject of intensive investigations over the past few decades. One very important group of receptors on the cell surface is the "integrin" receptors which bind to extracellular matrix (ECM) proteins. Because of integrin's importance in cellular growth, development, and morphology the role of integrin receptors in cellular transformation, malignant growth, and metastasis has received wide attention. In this article we report on the effect of all-trans-retinoic acid (ATRA) on (a) the integrin family of cell surface receptors, (b) collagenase enzyme activity, and (c) invasive potential in human cervical cancer (SiHa) cells. A comparative cell adhesion assay clearly showed that ATRA affects the cell surface integrin receptors against different ECM proteins in a dose- and time-dependent manner. The binding of SiHa cells to ECM proteins (fibronectin, vitronectin, laminin, collagen IV) was drastically reduced when cells were treated with ATRA at 10 microM for 96 h in culture. Interestingly, when ATRA-treated (10 microM, 96 h) SiHa cells were allowed to grow for 15 days in ATRA-free complete medium the binding of SiHa cells to fibronectin and vitronectin was inhibited, even after 15 days of drug withdrawal, whereas cell adhesion to laminin and collagen IV returned to normal within 3-7 days. The comparative immunoprecipitation of two cell surface integrin receptors (alpha5beta1 and alphavbeta3) shows the effect of ATRA on the expression of alpha5, alphav, and beta1 subunits. In ATRA-treated SiHa cells the cell surface expression of the alphav subunit (in alphavbeta3 receptor) is much less than in untreated SiHa cells. In the case of the alpha5beta1 integrin receptor ATRA treatment caused a significant reduction in the expression of both alpha5 and beta1 subunits on the cell surface. Comparative zymography clearly demonstrated the inhibitory effect of ATRA on collagenase enzyme activity. Interestingly, the effect was irreversible, even after 15 days of culture in ATRA-free medium. The assay of the invasive potential of ATRA-treated and untreated SiHa cells in Boyden's invasion chamber demonstrated that ATRA treatment (10 microM, 96 h) inhibits the invasive potential of SiHa cells. The effect was not reversible even after 15 days of culture in ATRA-free medium. In conclusion, our observations indicate that ATRA has an inhibitory effect on the expression of SiHa cell surface integrin receptors and collagenase enzyme activity. The effect of ATRA on cell surface integrin receptors and collagenase enzyme activity may affect the invasive potential of SiHa cells.

Cell Adhesion↗
PubMed1994

Multiple platelet surface receptors mediate platelet adhesion to surfaces coated with plasma proteins.

The interactions between platelets and plasma proteins previously shown to adhere to biomaterials were evaluated, using monoclonal antibodies (mAbs) against specific platelet surface glycoprotein (GP) receptors. Purified 51Cr-labeled human platelets in plasma-free medium were incubated with each of the following antibodies: mAb 10E5 [anti-GP IIb/IIIa; fibrinogen, von Willebrand factor (vWF), and fibronectin receptor]; mAb 6D1 (anti-GP Ib-IX; vWF receptor); mAb IV.3 (anti-Fc gamma RII; IgG receptor); polyclonal antiserum A108 or mAb BIIG4 (anti-GP Ic-IIa; fibronectin receptor). Antibody-treated platelets were added to microtiter wells coated with fibronectin, fibrinogen, vWF, IgG, vitronectin, albumin, or platelet-poor plasma (PPP). 51Cr-labeled platelet adhesion to matrix proteins was expressed as a percentage of that measured on PPP-coated surface. Platelets adhered to fibrinogen, fibronectin, vWF, or IgG immobilized on polystyrene. Limited binding to either vitronectin or albumin was detected. Binding to fibrinogen and IgG was blocked by mAb 10E5. Binding to IgG was also blocked by mAb IV.3. Binding to fibronectin, reduced in the presence of mAb 10E5, mAb BIIG4, or the polyclonal antiserum A108 alone, was further reduced by combined 10E5 and BIIG4 or 10E5 and A108. Neither mAb 10E5 nor 6D1 alone blocked adhesion to vWF; however, the combination of 10E5 and 6D1 significantly reduced platelet adhesion to this matrix. Finally, platelet adhesion to the plasma-coated surface was reduced by mAbs 10E5 and BIIG4. These results indicate that multiple adhesion receptors can mediate platelet adhesion to matrix proteins immobilized on surfaces.

Antibodies, Monoclonal↗
PubMed1994

The extracellular matrix of the fetal wound: hyaluronic acid controls lymphocyte adhesion.

Adhesive interactions between lymphocytes and components of the extracellular matrix (ECM) within a wound environment play a crucial role in determining the inflammatory response following tissue injury. In fetal wounds the extracellular matrix is composed predominantly of hyaluronic acid. Within this environment the inflammatory reaction as a result of injury is minimal. We propose that this lack of an inflammatory cell response in the fetal wound is due to the high levels of hyaluronic acid within the ECM and the inability of lymphocytes to adhere to this matrix component. Therefore, we examined the adhesive properties of fetal lymphocytes to fibronectin, vitronectin, collagen types I, III, IV, V, and hyaluronic acid--ECM components involved in fetal and adult wound environments. Fetal lymphocytes from both spleen and thymus demonstrated significant binding capabilities to fibronectin, vitronectin, and collagen types I and III. No intrinsic binding capabilities were detected to hyaluronic acid. Adhesion was not affected by the addition of IL-1, IFN-gamma, or phorbol dibutyrate. The inability of lymphocytes to adhere to hyaluronic acid helps to explain the lack of inflammation found in fetal wounds and serves to demonstrate the importance of ECM-lymphocyte interactions in determining the inflammatory response during fetal wound healing.

Animals↗
PubMed1997

Expression of two novel recombinant proteins from aortic adventitia (kappafibs) sharing amino acid sequences with cytomegalovirus.

We have recently purified and partially sequenced a microfibrillar protein from human aortic adventitia (aneurysm-associated antigenic protein, 40 kDa [AAAP-40]) that is immunoreactive with immunoglobulin (IgG) from the wall of abdominal aortic aneurysms (AAAs). It shares motifs with Ig kappa (which may act as a binding site for interaction with integrins), cytomegalovirus (which may be a molecular mimic in the pathogenesis of AAA), and vitronectin and the fibrinogens. A cDNA library was constructed from the aortic adventitia of a AAA. The library was screened with either rabbit anti-vitronectin antibody or rabbit anti-fibrinogen antibody. Positive plaques were purified and expressed in a strain of Escherichia coli. The clone sequences were analyzed. The expressed proteins were separated by SDS/PAGE and the immunoblots were probed with either AAA IgG or anti-human Ig kappa antibody. Experimental cell lines, transfected with the clones (clones 1 and 5), synthesized recombinant proteins (rAAAP-CL1 and rAAAP-CL5), detectable in Western immunoblots with AAA IgG. A prediction of the tertiary structure resembles well-characterized cell adhesion molecules. These findings suggest that there is a novel family of matrix proteins that may use immunoglobulin motifs as binding sites for cellular integrins and that there are matrix proteins in addition to AAAP-40 that may serve as autoantigens in the pathogenesis of AAA disease.

Amino Acid Sequence↗
PubMed2001

Association between integrin-dependent migration capacity of neural stem cells in vitro and anatomical repair following transplantation.

In previous transplantation studies using neural stem cell lines immortalized by the temperature-sensitive SV40 large T-antigen, we have shown that animals with experimental hippocampal lesions resulting from four vessel occlusion recover spatial memory functions more effectively when grafted with the MHP36 cell line than with the MHP15 cell line [Gray et al. (1999). Philos. Trans. R. Soc. London Biol. Sci. 354:1407-1421]. In the present study, we have investigated the cellular and molecular basis of these differences in repair capacity both in vivo and in vitro. Using the same model of hippocampal damage we have shown that following transplantation MHP36 cells migrate and align within the damaged CA1 of the ipsilateral hippocampus. MHP15 cells, in contrast, migrate in a more indiscriminate pattern that does not reflect the anatomy of the region. To analyze the migratory properties of these two cell lines in more detail, we performed migration assays at a nonpermissive temperature on the extracellular matrix substrates laminin, fibronectin, and vitronectin. These showed that MHP36 cells have a greater migration potential than the MHP15 cells. While the pattern of cell surface extracellular matrix receptors of the integrin family was identical in both cell lines, the different degrees of migration on vitronectin were both blocked by inhibitors of alphaV integrins. Differences in integrin signaling therefore contribute to the greater migration potential of the repairing MHP36 cell line.

Animals↗
PubMed1994

Purification and partial characterization of a glycoprotein from pea (Pisum sativum) with receptor activity for rhicadhesin, an attachment protein of Rhizobiaceae.

Attachment of Rhizobium and Agrobacterium bacteria to cells of their host plants is a two-step process. The first step, direct attachment of bacteria to the plant cell wall, is mediated by the bacterial protein rhicadhesin. A putative plant receptor molecule for rhicadhesin was purified from cell walls of pea roots using a bioassay based on suppression of rhicadhesin activity. This molecule appeared to be sensitive to treatments with pronase or glycosidase. Its isoelectric point is 6.4, and its apparent molecular mass was estimated to be 32 kDa before and 29 kDa after glycosidase treatment, as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and ultrafiltration. The sequence of the first 29 N-terminal amino acids was determined: A-D-A-D-A-L-Q-D-L-C(?)-V-A-D-Y-A-S-V-I-L- V-N-G-F-A-S-K(Q)-(P/Q)-(L)-(I). No homology with known proteins was found. In the course of this research project the extracellular matrix protein vitronectin was reported to inhibit attachment of A. tumefaciens to carrot cells [29]. A variety of adhesive proteins, including vitronectin, contain a common cell attachment determinant with the sequence R-G-D. Since we could not detect other cell wall components able to suppress rhicadhesin activity, and since an R-G-D containing hexapeptide was also active as a receptor, we speculate that the plant receptor for rhicadhesin is a glycoprotein containing an R-G-D attachment site.

Amino Acid Sequence↗
PubMed1996

Alterations in extracellular matrix components and integrins in patients with preeclamptic nephropathy.

The glomerular features of patients with preeclampsia consist of swelling of endothelial cells, subendothelial deposits of incompletely defined material, and thickening of the capillary walls. These abnormalities are thought to resolve in the postpartum period. The distribution of extracellular matrix (ECM) components and integrins was investigated in 10 such patients. Frozen sections and paraffin-embedded sections were stained with antibodies to type IV collagen, laminin (LN), fibronectin (FN), vitronectin (VN), tenascin (TN), fibronectin receptor (FNR), and vitronectin receptor (VNR). In preeclamptic nephropathy, the accumulation of type IV collagen, LN, FN, TN, and FNR was observed in the thickened capillary walls, particularly in the subendothelial layer and, to some extent, in the mesangium. However, deposits of VN were sparse and the distribution of VNR was similar to that in normal kidney. In segmental sclerotic lesions, the amounts of type IV collagen, LN, FN, VN, and TN were increased, whereas those of FNR and VNR were markedly decreased. These results suggest that the materials deposited in the subendothelial space consist of ECM components as well as of plasma-derived proteins, and that the deposition of ECM components and of FNR may be involved in the development and the reparative process of the characteristic glomerular lesions. The formation of sclerotic lesions was linked to the accumulation of ECM components, but not to an interaction with integrins.

Adult↗
PubMed1991

Histiocytosis X arising in Hodgkin's disease: immunophenotypic characterization with a panel of monoclonal antibodies.

This report describes the antigenic profile of the proliferating cells of pulmonary histiocytosis X (HX) in a patient treated with chemotherapy for Hodgkin's lymphoma; the association of pulmonary HX and Hodgkin's disease has rarely been described in the literature. The histopathological diagnosis of HX was confirmed with the aid of monoclonal antibodies (mAbs) to CD4, CD1a, and polyclonal serum anti S-100 protein. The phenotype of HX cells has been analysed using a panel of mAbs against HLA class I A, B, C monomorphic determinants, locus A and B, beta 2-microglobulin, HLA class II distinct monomorphic determinants, DP, DQ, DR, intercellular adhesion molecule-1 (ICAM-1) and vitronectin receptors. Our results indicate that HX cells express HLA class I and II, including locus A, locus B and DP, DQ, DR, like their normal counterpart (represented by Langerhans cells) and detectable levels of ICAM-1 but not vitronectin receptors. We would like to stress the possibility of the association of HX and Hodgkin's lymphoma extending the immunophenotypic profile of HX cells.

Adult↗
PubMedDate not supplied

Expression and role of integrins in adhesion of human colonic carcinoma cells to extracellular matrix components.

We have examined integrin expression and function in the human colon carcinoma cell line HT29, and in clonal sublines derived from the HT29 line. These cells express several different integrin subunits including beta 1, alpha 2, 3, 6 and alpha v, but do not express the classic alpha 5/beta 1 fibronectin receptor. Clonal variation in the pattern of integrin expression was quite limited. The profile of integrin expression correlates well with the adhesive behavior of HT29 cells. Thus the cells adhere well to vitronectin, laminin and type IV collagen, but not at all to fibronectin. Adhesion to collagen was completely blocked by an anti-beta 1 monoclonal antibody, indicating that beta 1 integrins mediate this process. Adhesion to laminin was strongly blocked by anti-beta 1 monoclonal or anti-beta 6 monoclonal, suggesting that the alpha 6/beta 1 complex functions in attachment to laminin; this was somewhat surprising since immunoprecipitation experiments indicate that most of the alpha 6 subunit seems to be associated with the beta 4 subunit. Despite their strong adherence to laminin, collagen and vitronectin, HT29 cells are not very motile and, in response to gradients of these proteins, do not migrate nearly as well as CHO cells tested under similar conditions. Since HT29 cells can undergo an enterocyte-like differentiation in glucose-free medium, we compared integrin expression in HT29 and its subclones during the process of differentiation. There was no correlation between the state of differentiation, as assessed by expression of brush-border hydrolases, and the level of expression of any of the integrin subunits measured. Thus the pattern of integrin expression in these colonic tumor cells seems to be a characteristic of the cell line, and is not readily modified by changes in cell growth or differentiation.

Carcinoma↗
PubMed1996

Mechanisms of colon cancer binding to substratum and cells.

Binding of colon cancer to extracellular matrix (ECM) proteins and mesenchymal cells that comprise the basement membrane is important in migration and metastasis. This study defines the conditions and surface structures necessary for adhesion of HT-29 cells to ECM proteins and cell monolayers. Binding began within minutes and peaked by 1 hr, with 80-95% of HT-29 cells binding to the ECM proteins, collagen IV, laminin, fibronectin, and vitronectin and 40-75% binding to monolayers of fibroblasts, smooth muscle cells, and HT-29 cells. Treating mesenchymal cells with the fibrogenic cytokines, IL-1, IL-4, or TNF-alpha, which increase production of ECM proteins, did not alter binding of HT-29 cells to these monolayers. Attachment of HT-29 cells to cell monolayers was inhibited by cytochalasin D and sodium azide, but not cycloheximide or neuraminidase. Attachment to ECM proteins, in contrast, was unaffected by any of these metabolic inhibitors but required certain divalent cations (Mg2+ and Mn2+ but not Ca2+). Antibody to the integrin beta 1, chain (CD29) eliminated binding to collagen and laminin but not to fibronectin, fibroblasts, and HT-29 monolayers. Antibody to the vitronectin receptor inhibited binding to fibronectin. Antibodies to integrin alpha 1-alpha 6 chains had no effect on any adhesion event. Three colon cancer cell lines were tested for expression of VLA antigens: alpha 2 and alpha 3 were detected on all three, alpha 1 and alpha 6 were variably expressed, while alpha 4 and alpha 5 were absent. This study demonstrates that several mechanisms account for tumor cell attachment to substratum and cells.

Basement Membrane↗
PubMed1995

Altered expression of extracellular matrix molecules and their receptors in chronic pancreatitis and pancreatic adenocarcinoma in comparison with normal pancreas.

The aim of this study was to elucidate the expression and distribution patterns of both integrins and extracellular matrix (ECM) molecules in chronic pancreatitis (CP) and pancreatic adenocarcinoma (PC) compared with normal pancreas (NP). Expression of nine alpha-subunits (alpha 2-alpha 6, alpha V, alpha L, alpha M, and alpha X), four beta-subunits (beta 1, beta 3-beta 5), and four ECM molecules (type IV collagen, laminin, fibronectin, and vitronectin) was investigated immunohistochemically. In CP, all integrins except alpha V showed nearly the same staining patterns compared with NP. Some acinar cells in CP expressed alpha V. Whereas alpha 2, alpha 3, and alpha 6 expression was stronger and diffuse, no alpha 5 expression was seen in PC. Basement membrane (BM) showed continuous staining in CP, whereas it showed discontinuous/absent staining in PC with antitype IV collagen, laminin, and vitronectin antibodies. Some carcinoma cells showed reverse correlation between alpha 2, alpha 3, and alpha 6 expression and type IV collagen and laminin expression. Fibronectin showed diffuse stromal expression in CP and PC. Some acinar cells or duct cells in CP carcinoma cells in PC showed intracellular VN expression. These results suggest that these integrins and ECM molecules are involved in inflammatory and malignant processes in pancreas.

Adenocarcinoma↗
PubMed1996

Tyrosine Phosphorylation of a ~30 kD Protein Precedes avb3 Integrin-signaled Endothelial Cell Spreading and Motility on Matrix Proteins.

A microvascular endothelial cell line (CD clone 4) isolated from murine lung adheres to and spreads well on fibronectin, vitronectin, and fibrinogen, but poorly on collagen type IV and laminin. Ligating cell surface av, b3, a4, a5, or b1 integrin receptors with monospecific antibodies promoted a dramatic cell spreading and motility on vitronectin or collagen IV. Antibodies directed to other adhesion molecules, including aIIb, PECAM-1, and P-selectin were ineffective. Ligation with monoclonal anti-av or -b3, but not -a4, -a5, or -b1 antibodies, induced a rapid, and dose-dependent tyrosine phosphorylation of a ~30 kD protein, which preceded CD clone 4 endothelial cell spreading and motility and was partially inhibited by genistein and completely inhibited by BAPTA. All other antibodies tested did not induce the tyrosine phosphorylation of the 30 kD protein as well as cell spreading and motility. The present results suggest that b1 and b3 integrins employ different biochemical mechanisms in signaling endothelial cell spreading and motility and that the tyrosine phosphorylation of the 30 kD protein (and probably other proteins) may play an important role in signaling b3 integrin-mediated endothelial cell interaction with other cells (e.g., tumor cells) and extracellular matrix.

Journal Article↗
PubMed1999

Thalidomide up-regulates prostate-specific antigen secretion from LNCaP cells.

Thalidomide has been shown to have species- and metabolic-dependent antiangiogenic activity in vitro and in vivo, suggesting its potential in treating human angiogenesis-dependent pathologies such as solid tumors. Based on promising preclinical studies, thalidomide has entered phase II clinical trials for prostate, brain, breast cancer, and Kaposi's sarcoma. However, the antiangiogenic mechanism of action is largely unresolved, as are its effects on tumor-associated gene expression, cytokine secretion, etc. We have investigated the effects of thalidomide on: 1) the secretion of prostate-specific antigen (PSA) in a human androgen-dependent prostate cell line; 2) growth and viability of human prostate cells; and 3) differential gene expression profiles of thalidomide-treated vs untreated human prostate cells. A human androgen-dependent prostate carcinoma cell line (LNCaP) and a human androgen-independent prostate carcinoma cell line (PC-3) were incubated with thalidomide 0.6, 6, or 60 microg/mL for 5-6 days. Secreted PSA from LNCaP cells was measured using a commercial enzyme-linked immunosorbant assay. Cell viability studies were conducted in both LNCaP and PC-3 cells using the same thalidomide concentrations. Furthermore, the differential gene expression of thalidomide-treated LNCaP cells was compared to that of untreated control cells using a commercially available human cancer cDNA expression array system. Thalidomide-treated LNCaP cells demonstrated increased PSA/cell levels at all concentrations tested compared to untreated control cells. Thalidomide demonstrated a cytostatic effect in LNCaP cells but had no appreciable effect on PC-3 cell viability compared to untreated control cells. Comparison of cDNA expression arrays hybridized with thalidomide-treated LNCaP cDNA probes suggests that thalidomide may up- or downregulate expression of angiogenesis-related genes, i.e., vitronectin, but these differential effects require further verification. Thalidomide over a range of doses has demonstrated nontoxic, cytostatic activity in LNCaP cells and significant upregulation of LNCaP cell PSA secretion in vitro. Furthermore, preliminary data from cDNA nucleic acid arrays of thalidomide-treated LNCaP cells suggest that thalidomide upregulates a potential angiogenic modulatory protein, the vitronectin precursor, which may eventually link thalidomide's antiangiogenic activity with modulation of angiogenic vascular integrin pathways.

Antineoplastic Agents↗