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PubMed1995

Alpha v and alpha 3 integrin subunits are associated with myofibrils during myofibrillogenesis.

The development of the myofibrillar apparatus in skeletal muscle is a process in which transmembrane linkages with adhesion molecules are implicated. Integrins are one class of transmembrane adhesion receptors which appear to mediate these interactions. Two prominent linkages are at the myotendinous junction (MTJ), which resides at the ends of the cell and connects myofibrils to the tendon, and the costameres, which encircle the girth of the cell and connect the Z-disks to the sarcolemma. In this study we report that the alpha v integrin subunit is a prominent component of the costamere. The alpha v subunit is present initially on developing myotubes in a diffuse staining pattern with some concentration along nascent myofibrils. However, it appears in a striated pattern at the costamere and inconsistently at the M-line following the striation of alpha-actinin and titin but before that of desmin. Its recruitment to preformed striation suggests that it is incorporated into a pre-existing structure. The presence of alpha v in the costamere points to a role in lateral myofibrillar anchorage. In addition, we find that the alpha 3 subunit is transiently associated with myofibrils along portions of their lengths and at their ends during myofibrillogenesis. The alpha 3 subunit staining shows a novel localization and junctional structure. As myofibrils become striated the alpha 3 integrin dissociates from the localized pattern and becomes diffuse. This suggests a possible role in the stabilization of nascent myofibrils prior to striation. Antibody-induced perturbation of adhesion mediated by the integrin beta 1 subunit in developing myotubes inhibits assembly of the sarcomeric architecture.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1997

Fibronectin provides a conduit for fibroblast transmigration from collagenous stroma into fibrin clot provisional matrix.

After injury, the wound space is filled with a fibrin/fibronectin clot containing growth factors released by platelets and monocytes. In response to these factors, fibroblasts migrate into the fibrin clot and contribute to the formation of granulation tissue. The functional mechanisms allowing fibroblasts to leave the collagenous matrix of normal connective tissue and invade the provisional matrix of the fibrin clot have not been fully defined. To investigate these mechanisms we established a new in vitro model which simulates specific aspects of early wound healing, that is, the migration of fibroblasts from a three-dimensional collagen matrix into a fibrin clot. This transmigration could be induced by physiological concentrations of platelet releasate or platelet-derived growth factor BB (PDGF-BB) in a concentration-dependent manner. At 24 hours irradiated fibroblasts invaded the fibrin gel almost as well as non-irradiated cells, indicating that transmigration was independent of proliferation. Plasminogen and its activators appear to be necessary for invasion of the fibrin clot since protease inhibitors decreased the amount of migration. These serine proteases, however, were not necessary for exit from the collagen gel as fibroblasts migrated out of the collagen gel onto a surface coated with fibrin fibrils even in the presence of inhibitors. Removal of fibronectin (FN) from either the collagen gel or the fibrin gel markedly decreased the number of migrating cells, suggesting that FN provides a conduit for transmigration. Cell movement in the in vitro model was inhibited by RGD peptide, and by monoclonal antibodies against the subunits of the alpha5 beta1 and alpha v beta3 integrin receptor. Thus, the functional requirements for fibroblast transmigration from collagen-rich to fibrin-rich matrices, such as occurs in early wound healing, have been partially defined using an in vitro paradigm of this important biologic process.

Antigens, CD↗
PubMed2001

Regulation of human lung fibroblast phenotype and function by vitronectin and vitronectin integrins.

Myofibroblasts, characterised by high expression of alpha-smooth muscle actin (alpha-SMA), are important and transient cells in normal wound healing but are found in increased number in various pathological conditions of the lung including asthma and pulmonary fibrosis. The mechanisms that regulate the myofibroblast phenotype are unknown but are likely to involve signals from the extracellular matrix transmitted via specific integrins. Vitronectin is a glycoprotein released during inflammation and has been shown to regulate the phenotype of vascular smooth muscle cells via alpha v and beta 1 integrins. In the current study we have examined whether vitronectin influences the phenotype and function of normal human lung fibroblasts (HFL-1). Incubation of HFL-1 cells with vitronectin induced a concentration-dependent reduction in alpha-SMA expression. By contrast, function-blocking monoclonal antibodies to the vitronectin integrins alpha v, beta 1, alpha v beta 3 and alpha v beta 5 induced the expression of alpha-SMA and its organization into stress fibers. Expression of alpha-SMA induced by all function-blocking monoclonal antibodies was abrogated by inhibition of protein kinase C and phosphatidylinositol-3 kinase, but the effects of inhibition of other signalling pathways was integrin dependent. Exposure to other extracellular matrix proteins such as fibronectin, collagen or their integrins did not influence expression of alpha-SMA. The expression and organization of alpha-SMA induced by exposure to function-blocking antibodies was translated into an augmented capacity of HFL-1 cells to contract fibroblast populated collagen gels. By contrast, contraction of collagen gels following incubation with vitronectin was not significantly different to control. This study has shown that vitronectin influences the phenotype and behaviour of HFL-1 cells by downregulating the expression of alpha-SMA and reducing their contractile ability. By contrast, occupancy of specific integrins by function-blocking antibodies upregulated the expression of alpha-SMA and induced the formation of functional stress fibers capable of contracting collagen gels. These results suggest that vitronectin modulates the fibroblast-myofibroblast phenotype, implying an important role in the remodelling process during lung development or response to injury.

Actins↗
PubMed2000

Suppression of integrin alpha(v) expression by endothelin-1 in vascular smooth muscle cells.

Both integrins and endothelins (ETs) are known to play important roles in vascular remodeling via proliferation, apoptosis, and migration of vascular smooth muscle cells (VSMCs), whose dysfunctions have been implicated in the pathogenesis of end-organ damage associated with hypertension and arteriosclerosis. However, whether there is any interaction between endothelin-1 (ET-1) and integrins remains unknown. Therefore, the aim of the present study was to elucidate whether ET-1 regulates the expression of integrin alpha(v) in rat VSMCs. ET-1 dose- and time-dependently suppressed the integrin alpha(v) messenger RNA (mRNA) transcripts, as quantified by a real-time quantitative polymerase chain reaction (PCR) method, and decreased the transcriptional activity of integrin alpha(v) gene, as demonstrated by integrin alpha(v)-luciferase assay. The inhibitory effect of ET-1 on integrin alpha(v) gene expression was abrogated by an ETA receptor antagonist (BQ123) but not by an ET(B) receptor antagonist (BQ788). ET-1 also suppressed the cell surface expression of integrin alpha(v)beta5 and the adhesion to vitronectin, but not to fibronectin. These results demonstrate that the adhesion of vitronectin to rat VSMCs is inhibited by ET-1 via the ET(A) receptors by suppressing integrin alpha(v) gene transcription, suggesting that ET-1 is involved in regulation of vascular integrin alpha(v) gene expression.

Animals↗
PubMed2003

Induction of apoptosis in vascular cells by plasminogen activator inhibitor-1 and high molecular weight kininogen correlates with their anti-adhesive properties.

Plasminogen activator inhibitor-1 (PAI-1) and two-chain high molecular weight kininogen (HKa) exert anti-adhesive properties in vitronectin-dependent cell adhesion. Here, the hypothesis was tested that these anti-adhesive components promote apoptosis in vascular cells. PAI-1 or HKa induced a 2- to 3-fold increase in apoptosis of human umbilical-vein endothelial cells (HUVEC) and vascular smooth muscle cells (VSMC) adherent to vitronectin, as determined by annexin V-FACS assay, similar to alphav-integrin inhibitor cyclo-(Arg-Gly-Asp-D-Phe-Val)-peptide (cRGDfV). Apoptosis occurred after 12 h incubation and was attributable to caspase 3 activation that in turn induced DNA fragmentation. Induction of apoptosis strongly correlated with the anti-adhesive effect of PAI-1 and HKa on these cells. In contrast, PAI-1 and HKa did not affect fibronectin-dependent adhesion or cell survival. uPA did not influence apoptosis in vitronectin- or fibronectin-adherent cells. In atherosclerotic vessel sections, congruent distribution of vitronectin, PAI-1, HK, and of components of the urokinase plasminogen activator/receptor system with apoptotic cells lining foam cell lesions was demonstrated by immunostaining. These results indicate that inhibition of vitronectin-dependent cell adhesion through PAI-1 and HKa correlates with apoptosis induction in vascular cells mediated through the caspase 3 pathway. Co-distribution of apoptosis with plasminogen activation system components in atherosclerosis exemplifies the significance of anti-adhesive mechanisms and apoptosis for tissue remodeling, such as in neointima development.

Apoptosis↗
PubMed1996

PDGF-B producing cells and PDGF-B gene expression in normal gingival and cyclosporine A-induced gingival overgrowth.

It has been proposed that healthy gingiva is in a continuous state of wound repair. Thus, one might expect to find cells in normal gingiva producing growth factors associated with wound healing such as platelet-derived growth factor B chain (PDGF-B). One might also expect to find increased numbers of these cells or increased amounts of these growth factors in conditions which involve increased tissue volume such as drug-induced gingival overgrowth (DGO). The purpose of this study was to quantify PDGF-B gene expression and identify cells producing PDGF-B in normal gingiva and DGO. Cyclosporine A (CSA) was selected as a prototype of the overgrowth condition. Twelve patients with clinical CSA DGO and 12 patients with no DGO or history of drugs known to cause DGO were selected for study. Frozen sections of gingival specimens from these patients were subjected to in situ hybridization for PDGF-B mRNA. Positive cells were counted and expressed as mean +/- SEM cells/mm2 of lamina propria. Morphometric analysis revealed 6.2 +/- 1.9 cells/mm2 for control gingiva and 10.3 +/- 3.4 cells/mm2 for CSA DGO samples. There was no statistically significant difference between groups. PDGF-B gene expression was measured in these cells and expressed as mean +/- SEM silver grains/cells. There was a significant upregulation of PDGF-B gene expression in cells from the CSA DGO group (39.5 +/- 14.7 silver grains/cell for normal gingiva vs. 255.3 +/- 77.1 silver grains/cell for CSA DGO samples; P < 0.001). The presence of PDGF-B in these cells was confirmed in all cases by immunocytochemical localization. Additionally, PDGF-B producing cells were identified as macrophages in sections taken from an additional patient with CSA DGO by double immunofluorescence labeling of the CD51 membrane marker for macrophages and intracellular PDGF-B. These findings are consistent with the concept that healthy gingiva is in a continuous state of wound repair and support the hypothesis that CSA DGO is associated with enhanced macrophage PDGF-B gene expression rather than an increase in the number of PDGF-B producing macrophages.

Antigens, CD↗
PubMed1996

Cyclosporine A upregulates platelet-derived growth factor B chain in hyperplastic human gingiva.

Cyclosporine A (CSA) is a widely used immunosuppressant for transplant patients and is also used for the treatment of a wide variety of systemic diseases with immunologic components. A prominent side effect of CSA administration is gingival overgrowth (hyperplasia). It has been postulated that CSA alters fibroblast activity through effects on various growth factors/cytokines. However, as yet, data concerning the molecular mechanisms involved in pathologic connective tissue proliferation are preliminary in nature. Our previous investigations concerning phenytoin-induced effects on platelet-derived growth factor B (PDGF-B) gene expression have demonstrated that other drugs which cause gingival overgrowth can upregulate macrophage PDGF-B gene expression in vitro and in vivo. The purpose of the present study was to evaluate PDGF-B gene expression in gingival tissues of patients receiving CSA therapy and exhibiting gingival overgrowth to determine if similar PDGF-B upregulation occurs in response to CSA and to identify PDGF-B producing cells in these tissues. Quantitative competitive reverse transcription polymerase chain reaction (QC-RTPCR) techniques were utilized to measure PDGF-B mRNA levels in CSA overgrowth patients and normal controls (N = 6/group). Results were expressed as mean +/- mRNA copy number and tested for significance using unpaired t-tests. Gingival samples were harvested (standardized for local inflammation at the sample site), total RNA was extracted, and QC-RTPCR was performed using specific PDGF-B primers and a corresponding competitive internal standard. CSA-treated patients exhibiting gingival overgrowth demonstrated approximately 48-fold increase in PDGF-B mRNA (7667.1 +/- 477.4 copies for CSA patients vs. 158.2 +/- 37.1 copies for controls; P < 0.001). Additionally, dual fluorescence immunohistochemistry for mature macrophage marker antigen (CD51) and intracellular PDGF-B was utilized to identify and localize PDGF-B producing cells were demonstrated to be macrophages distributed in a non-uniform manner throughout the papillary connective tissue. These results further support the hypothesis that the molecular mechanisms responsible for drug-induced gingival overgrowth may involve upregulation of PDGF-B macrophage gene expression. We continue to investigated specific CSA-induced alterations of macrophage PDGF-B gene expression in vitro and in vivo.

Adult↗
PubMed1998

The response of periodontal ligament cells to fibronectin.

Fibronectin (fn) is an extracellular matrix (ECM) molecule important in cell adhesion and migration and in wound healing. It is also likely important in periodontal ligament (PDL) cell-ECM interactions, and thus in regenerating periodontal tissues. In this study we characterized PDL cells and their interactions with FN, testing different PDL cell isolates taken from healthy and diseased conditions. PDL cells were characterized by their morphology, integrin profile, motility, and bone nodule formation. Cells were then assayed for adhesion, proliferation, and chemotaxis in response to FN or FN fragments. Cell isolates were morphologically heterogeneous and fibroblastic, had a normal-appearing actin cytoskeleton and a wide range of migration potentials, and formed bone-like nodules in vitro. They expressed alpha5, beta1, alpha v, and alpha4 integrin subunits, known receptors for FN, and in fact they bound FN preferentially at 5 and 10 microg/ml. Intact FN induced greater PDL cell proliferation and chemotaxis than did FN fragments (120-kDa cell-binding, 60-kDa heparin-binding, and 45-kDa collagen-binding). PDL cells harvested from diseased and healthy conditions were no different on the basis of these assays. These data demonstrate that PDL cells are a mixed population of fibroblastic cells, capable of forming a mineralized matrix. They also suggest that maximal proliferation and chemotaxis require specific FN domains that are present on the intact molecule but not its fragments.

Actins↗
PubMed2001

The dento-epithelial junction: cell adhesion by type I hemidesmosomes in the absence of a true basal lamina.

BACKGROUND: The junctional epithelium (JE) is a unique structure that makes contact with both a non-renewable hard tooth surface and with a basement membrane (BM) facing the connective tissue. Ultrastructurally, this attachment occurs through hemidesmosomes (HD) and a basal lamina-like extracellular matrix which, on the tooth side, is termed the internal basal lamina. In this study we investigated the expression of basal cell markers in the tooth-facing (TF) cells of JE. METHODS: Samples of healthy marginal gingiva were removed by careful dissection. The expression of laminin-5 was used to indicate TF cell preservation in double immunofluorescence labeling and confocal laser scanning microscopy. RESULTS: The results show that integrin alpha6beta4 and laminin-5 colocalize unequivocally in the TF cells. The results also show the specific expression of the basal cytokeratin 14 and the alpha(v) integrin subunit in the TF cells. All 3 major hemidesmosomal components BP180, BP230, and HD1 antigen are likewise present. On the other hand, type IV collagen, laminin-1/10, type VII collagen, and the BM proteoglycan perlecan are all absent from the dento-epithelial junction. CONCLUSIONS: The results indicate that the epithelium-tooth interface is a unique structure wherein epithelial cells adhere by means of bona fide hemidesmosomes to an epithelium-derived extracellular matrix lacking most of the common BM components. Moreover, TF cells differ from connective tissue facing (CTF) cells, not only by their cell surface molecules and their production of extracellular matrix, but also by their cytoskeletal architecture.

Adolescent↗
PubMed2000

Modulation of heart fibroblast migration and collagen gel contraction by IGF-I.

Dynamic interactions between cells and the extracellular matrix are essential in the regulation of a number of cellular processes including migration, adhesion, proliferation and differentiation. A variety of factors have been identified which modulate these interactions including transforming growth factor-beta, platelet-derived growth factor and others. Insulin-like growth factors have been shown to regulate collagen production by heart fibroblasts; however, the effects of this growth factor on the interactions of heart fibroblasts with the extracellular matrix have not been examined. The present studies were carried out to determine the effects of IGF-I on the ability of fibroblasts to interact with the extracellular matrix and to begin to determine the mechanisms of this response. These experiments illustrate that IGF-I treatment results in increased migration, collagen reorganization and gel contraction by heart fibroblasts. IGF-I has been shown to activate both the mitogen-activated protein kinase and phophatidylinositol-3 kinase pathways in isolated cells. Experiments with pharmacological antagonists of these pathways indicate that the mitogen-activated protein kinase pathway is essential for IGF-I stimulated collagen gel contraction by fibroblasts. These studies illustrate that IGF-I modulates the ability of fibroblasts to interact with the collagen matrix and that activation of multiple signaling pathways by IGF-I may produce distinct downstream responses in these cells.

Animals↗
PubMed1995

Differential mRNA regulation of integrin subunits alpha V, beta 1, beta 3, and beta 5 during mouse embryonic organogenesis.

Cell interactions with extracellular matrices play important roles in morphogenetic processes during embryonic development. Extracellular matrix receptors of the integrin family have been implicated in these steps. Recent studies indicate that a variety of integrins can be differentially expressed during development, consistent with diverse roles for integrins in embryogenesis. The present study compares the expression patterns of several major members of the alpha V integrin subfamily, focusing on mRNA expression of alpha V, beta 1, beta 3, and beta 5 subunits during mouse embryonic organogenesis using Northern blot analysis and in situ hybridization. The alpha V and beta 1 subunits showed widespread tissue expression, although most tissues expressed alpha V at relatively low or basal levels. The mRNA for beta 5 was also expressed in a variety of embryonic organs and showed unusual localization patterns in certain organs. Striking, high-level expression of beta 5 transcripts was detected in the ependymal layer of the central nervous system, glomeruli of the kidney, epicardial region of the heart, and in the tooth germs, suggesting specific functions for this molecule during morphogenetic events in these organs. In contrast, few beta 3 transcripts were expressed during mid-gestation mouse embryogenesis except in megakaryocytes within the embryonic liver. These observations of differing expression spectra suggest that members of the alpha V integrin subfamily have distinct roles and also suggests that they have different transcriptional regulation. The beta 5 integrin is unique in its degree of tissue-specific mRNA regulation associated with morphogenesis of embryonic organs.

Animals↗
PubMed1996

Identification of a new biological function for the integrin alpha v beta 3: initiation of fibronectin matrix assembly.

Fibronectin matrix assembly is a complex cellular process initiated by specific fibronectin-binding cell surface receptors. Although the integrin alpha 5 beta 1 has been implicated in the assembly of fibronectin matrices, fibroblastic cells derived from alpha 5 integrin null mutant embryos assemble a fibronectin matrix. Thus, alternative receptors must support this process. Although the platelet integrin alpha IIb beta 3 supports fibronectin matrix assembly, its expression is restricted to platelets. We report that alpha v beta 3 integrin, a fibronectin receptor expressed on many cell types provides an alternative pathway for the assembly of soluble fibronectin into the extracellular matrix. This process occurs independent of alpha 5 beta 1, is also modulated by activation, and the resulting matrix is biochemically indistinguishable from that assembled under the control of alpha 5 beta 1. Matrix assembly requires binding to the RGD site in the 10th type III repeat of fibronectin, as well as the participation of the amino-terminal matrix assembly domain. The participation of two distinct integrins in fibronectin matrix assembly suggests a model for the involvement of integrins in a dual system of extracellular matrix assembly and recognition controlled by intracellular activation of extracellular receptors.

Animals↗
PubMed2000

In vivo roles of integrins during leukocyte development and traffic: insights from the analysis of mice chimeric for alpha 5, alpha v, and alpha 4 integrins.

Mice chimeric for integrins alpha(5), alpha(V), or alpha(4) were used to dissect the in vivo roles of these adhesion receptors during leukocyte development and traffic. No major defects were observed in the development of lymphocytes, monocytes, or granulocytes or in the traffic of lymphocytes to different lymphoid organs in the absence of alpha(5) or alpha(V) integrins. However, in agreement with previous reports, the absence of alpha(4) integrins produced major defects in development of lymphoid and myeloid lineages and a specific defect in homing of lymphocytes to Peyer's patches. In contrast, the alpha(4) integrin subunit is not essential for localization of T lymphocytes into intraepithelial and lamina propria compartments in the gut, whereas one of the partners of alpha(4), the beta(7) chain, has been shown to be essential. However, alpha(4)-deficient T lymphocytes cannot migrate properly during the inflammatory response induced by thioglycolate injection into the peritoneum. Finally, in vitro proliferation and activation of lymphocytes deficient for alpha(5), alpha(V), or alpha(4) integrins upon stimulation with different stimuli were similar to those seen in controls. These results show that integrins play distinct roles during in vivo leukocyte development and traffic.

Animals↗
PubMed2001

Nonphlogistic clearance of late apoptotic neutrophils by macrophages: efficient phagocytosis independent of beta 2 integrins.

Neutrophils undergo constitutive death by apoptosis, leading to safe nonphlogistic phagocytosis and clearance by macrophages. Recent work has shown that before secondary necrosis, neutrophils exhibiting classical features of apoptosis can progress to a morphologically defined late apoptotic state. However, whether such neutrophils could be safely cleared was unknown. We now report that human late apoptotic neutrophils could be purified from cultured neutrophil populations undergoing constitutive death and were subsequently ingested by human monocyte-derived macrophages by serum-independent mechanisms that did not trigger the release of IL-8 or TNF-alpha. Such ingestion was specifically inhibited by Abs to thrombospondin-1 and the alpha(v)beta(3) vitronectin receptor. Murine bone marrow-derived macrophage phagocytosis of late and early apoptotic neutrophils occurred by similar mechanisms, proceeding with the same efficiency as that observed for wild-type controls when macrophages from [alpha(m)](-/-) or [beta(2)](-/-) mice were used. We conclude that specific nonphlogistic, beta(2) integrin-independent mechanisms involving thrombospondin-1 and alpha(v)beta(3) allow macrophages to ingest late apoptotic neutrophils without eliciting inflammatory cytokine secretion.

Animals↗
PubMed2003

The extracellular matrix and cytokines regulate microglial integrin expression and activation.

Microglia are the primary immune effector cells resident within the CNS, whose activation into migratory, phagocytic cells is associated with increased expression of cell adhesion molecules of the integrin family. To determine which specific factors are important regulators of microglial activation and integrin expression, we have examined the influence of individual cytokines and extracellular matrix (ECM) substrates by quantifying cell surface expression of MHC and individual integrins by flow cytometry. We found that the proinflammatory cytokines TNF and IFN-alpha promoted microglial activation, as assessed by amoeboid morphology and increased expression of MHC class I, and also increased expression of the alpha(4)beta(1) and Mac-1 integrins. In contrast, TGF-beta1 had the opposite effect and was dominant over the other cytokines. Furthermore, the ECM substrates fibronectin and vitronectin, but not laminin, also promoted microglial activation and increased expression of the alpha(4)beta(1), alpha(5)beta(1) and Mac-1 integrins, but significantly, the influence of fibronectin and vitronectin was not diminished by TGF-beta1. Taken together, this work suggests that, in addition to cytokines, the ECM represents an important regulatory influence on microglial activity. Specifically, it implies that increases in the local availability of fibronectin or vitronectin, as a result of blood-brain barrier breakdown or increased expression in different pathological states of the CNS, could induce microglial activation and increased expression of integrins.

Animals↗
PubMedDate not supplied

Novel small molecule alpha v integrin antagonists: comparative anti-cancer efficacy with known angiogenesis inhibitors.

Recent evidence supports the involvement of integrins in angiogenesis: blockade of alpha v beta 3 and alpha v beta 5 integrins disrupts angiogenesis leading to decreased blood vessel formation and hence decreased tumor growth. We hypothesized that av antagonists could inhibit tumor growth in tumor cells devoid of alpha v beta 3 integrins. We evaluated SM256 and SD983, novel small molecules that are specific av antagonists in mouse models of angiogenesis and tumorigenesis, and compared them with standards: TNP470, a fumagillin analog in the clinic, and flavopiridol, a cell cycle kinase inhibitor. In vitro SM256 was a selective alpha v beta 3 inhibitor with an IC50 = 4nM, and the affinity of SD983 against the mouse endothelial alpha v beta 3 integrin yielded an IC50 = 2nM and an IC50 = 54nM against alpha v beta 5. In the mouse Matrigel model of angiogenesis SM256 decreased blood vessel formation (hemoglobin content) with an ED50 = 0.055 ug/kg/day, tenfold more potent than TNP470. SG545, an ester of SD983, decreased blood vessel formation with an ED50 = 6 ug/kg/day, while flavopiridol ED50 = 18 ug/kg/day. In the mouse xenograft model, using human colon carcinoma RKO cells that do not express alpha v beta 3 but express alpha v beta 5, tumor growth was inhibited by SG545 (10 mg/kg/day) and flavopiridol (5 mg/kg/every other day) 40% and 70%, respectively (p < 0.05). Although the proliferative index (measured by BrdU incorporation) was not significantly changed with SM256, SG545 or flavopiridol (29-32%), the apoptotic index increased significantly (p < 0.05) in the SM256 and SG545-treated groups (2.3-2.7%) compared with controls (1.1%), suggesting increased cell death contributed to decreased tumor volumes. Neovascularization decreased with SM256 and SG545 treatment. The data demonstrate that potent selective av antagonist can target endothelial cells, tumor cells, inhibit angiogenesis and inhibit tumor growth.

Animals↗
PubMed1999

Fibronectin upregulates gelatinase B (MMP-9) and induces coordinated expression of gelatinase A (MMP-2) and its activator MT1-MMP (MMP-14) by human T lymphocyte cell lines. A process repressed through RAS/MAP kinase signaling pathways.

T-lymphocyte migration into tissues requires focal degradation of the basement membrane. In this study, we show that transient adherence to fibronectin induces the production of activated forms of matrix metalloproteinase-2 (MMP-2) and MMP-9, as well as downregulation of tissue inhibitor of metalloproteinase-2 (TIMP-2) by T-cell lines. MMP-2 activation was likely achieved by inducing a coordinated expression of membrane-type matrix metalloproteinase-1 (MMP-14), a major activator of MMP-2. Blocking monoclonal antibodies against alpha4, alpha5, and alphav integrins strongly reduced MMP-2 and MMP-9 production induced by fibronectin. Disrupting actin cytoskeleton organization by cytochalasin D strongly enhanced fibronectin-induced MMP-2 and MMP-9 expression. Inhibiting Src tyrosine kinases with herbimycin A reduced MMP-2 and MMP-9 production with no effect on cell attachment. By contrast, G-protein inhibition by pertussis toxin, or transfection with a dominant negative mutant of Ha-Ras strongly increased fibronectin-induced MMP-2 and MMP-9. Inhibition of PI3 kinase, MAPkinase (MEK1), or p38 MAPkinase by wortmannin, PD 98059, or SB 202190, respectively, strongly promoted fibronectin-induced MMP2 and MMP-9. Cells at high density lost their ability to synthesize MMP-2 and MMP-9 in response to fibronectin and MMP expression was restored by transfection with a dominant-negative mutant of Ha-Ras or by treatment with wortmannin, PD 98059, or SB 202190. Our findings suggest that adhesion to fibronectin transduces both stimulatory (through Src-type tyrosin kinases) and inhibitory signals (through Ras/MAPKinase signaling pathways) for MMP-2 and MMP-9 expression by T lymphocytes and that their relative predominance is regulated by additional stimuli related to cell adhesion, motility, and growth.

Androstadienes↗
PubMed1999

Growth suppression of human ovarian cancer cells by adenovirus-mediated transfer of the PTEN gene.

A tumor suppressor gene on chromosome 10q23, PTEN, encodes a phosphatidylinositol phosphatase that antagonizes activation of the phosphatidylinositol 3'-kinase-mediated pathway involved in cell growth. A gene encoding the catalytic subunit of phosphatidylinositol 3'-kinase (PIK3CA) is frequently activated in ovarian cancers; therefore, overexpression of the PTEN product through gene transfer might be an effective strategy for treating ovarian cancers. To test the potential for this type of gene therapy, we constructed a recombinant adenovirus encoding wild-type PTEN and examined its effects on nine cell lines derived from human ovarian carcinomas. Transduction of the PTEN gene significantly inhibited growth of six of these cell lines compared with infection with virus alone, and the degree of inhibition correlated with the efficiency of gene transfer as determined by beta-galactosidase assay. Results of flow cytometry suggested that the observed effects were mediated by two mechanisms, apoptosis and/or arrest in the G1 phase of the cell cycle, and that high adenoviral transduction efficiency of cells was associated with induction of apoptosis. We also found that the level of transcription of Integrin alpha(v) in ovarian cancer cells correlated with the efficiency of transduction (P = 0.014) and with the degree of growth inhibition after PTEN gene transfer (P = 0.009). These findings carry significant implications for adenovirus vector-based PTEN gene therapies for ovarian cancers.

Adenoviridae↗