Osteopontin Deficiency in Rat Vascular Smooth Muscle Cells is Associated with an Inability to Adhere to Collagen and Increased Apoptosis

Department of Pediatrics, The Mount Sinai School of Medicine, New York, New York 10029, USA.
Laboratory Investigation (Impact Factor: 3.68). 12/2000; 80(11):1603-15. DOI: 10.1038/labinvest.3780171
Source: PubMed


Osteopontin (OPN) is an extracellular matrix protein that has been implicated in vascular smooth muscle cell (VSMC) adhesion. We have previously described the generation of OPN-deficient VSMC that displayed altered adhesion to collagen. We have examined further the causes and consequences of this altered adhesion. OPN-deficiency was associated with a significant reduction in surface expression of alpha1 and beta1 integrins (mean fluorescence intensity alpha1: OPN-deficient 0.135+/-0.04 vs. control 0.313+/-0.05, p < 0.0001; beta1: OPN-deficient 0.398+/-0.09 vs. control 0.570+/-0.05, p < 0.004). Treatment of normal VSMC with antibody to alpha1 recapitulated the adhesion defect. OPN-deficient cells without collagen exposure had an apoptotic fraction of 1.9%, which increased to 95.7% after 24 hours exposure to collagen. Exogenous OPN added to cultures within 15 minutes of plating restored normal cell adhesion, but did not prevent cells from undergoing apoptosis. Normal VSMC had no detectable apoptosis after 24 hours incubation in suspension, whereas OPN-deficient cells had an apoptotic fraction of 37.5% when incubated in suspension under the same conditions. The data suggest that OPN-deficient VSMC have two distinct abnormalities: an alpha1beta1-mediated inability to adhere normally to collagen and an increased propensity for apoptosis.

Download full-text


Available from: Andrea S Weintraub, Feb 09, 2015
  • Source
    • "SMCs display remarkable plasticity and can undergo changes in phenotype , a process referred to as phenotypic switching, and often exist as a continuum of phenotypes consisting of contractile SMCs which have a complete functional contractile apparatus to the other extreme referred to as proliferating " synthetic " SMCs15161718. Although originally isolated from bone, OPN has been identified in many tissues, including vascular SMCs505152535455565758. OPN is a biomarker of vascular SMC phenotypic switching and is strongly expressed by synthetic SMCs [59], suggesting that we had some degree of synthetic SMC phenotypes present. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Background aims. Mesenchymal stromal cells (MSCs) have great potential for use in cell-based therapies for restoration of structure and function of many tissue types including smooth muscle. Methods. We compared proliferation, immunophenotype, differentiation capability and gene expression of bone marrow–derived MSCs expanded in different media containing human serum, plasma and platelet lysate in combination with commonly used protocols for myogenic, osteogenic, chondrogenic and adipogenic differentiation. Moreover, we developed a xenogenic-free protocol for myogenic differentiation of MSCs. Results. Expansion of MSCs in media complemented with serum, serum + platelet lysate or plasma + platelet lysate were multipotent because they differentiated toward four mesenchymal (myogenic, osteogenic, chondrogenic, adipogenic) lineages. Addition of platelet lysate to expansion media increased the proliferation of MSCs and their expression of CD146. Incubation of MSCs in medium containing human serum or plasma plus 5% human platelet lysate in combination with smooth muscle cell (SMC)-inducing growth factors TGFβ1, PDGF and ascorbic acid induced high expression of ACTA2, TAGLN, CNN1 and/or MYH11 contractile SMC markers. Osteogenic, adipogenic and chondrogenic differentiations served as controls. Discussion. Our study provides novel data on the myogenic differentiation potential of human MSCs toward the SMC lineage using different xenogenic-free cell culture expansion media in combination with distinct differentiation medium compositions. We show that the choice of expansion medium significantly influences the differentiation potential of human MSCs toward the smooth muscle cell, as well as osteogenic, adipogenic and chondrogenic lineages.These results can aid in designing studies using MSCs for tissue-specific therapeutic applications.
    Full-text · Article · Mar 2016 · Cytotherapy
  • Source
    • "Binding of OPN to the above receptors on tumor cells triggers downstream signaling pathways including Ras, Akt, MAPK, Src, FAK and NF-KB [1] that collectively lead to the following in tumor cells: i) invasion to ECM (extracellular matrix) mainly via upregulation of MMPs [19] (matrix metalloproteinases) and uPAs [20] (urokinase plasminogen activator) by OPN; ii) increased migration and adhesion of tumor cells [21]; iii) inhibition of cell death likely through upregulation of anti-apoptosis mediators such as GAS6 [22]; and iv) development of pre-metastatic niche [23]. Additionally, tumor stroma such as endothelial cells [18] and immune infiltrating cells [24,25] (particularly monocytes) express OPN receptors. Angiogenesis is proven to be a critical component of tumor mass by supplying oxygen and nutrients for cancer cells [26]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Osteopontin (OPN), also known as SPP1 (secreted phosphoprotein), is an integrin binding glyco-phosphoprotein produced by a variety of tissues. In cancer patients expression of OPN has been associated with poor prognosis in several tumor types including breast, lung, and colorectal cancers. Despite wide expression in tumor cells and stroma, there is limited evidence supporting role of OPN in tumor progression and metastasis. Using phage display technology we identified a high affinity anti-OPN monoclonal antibody (hereafter AOM1). The binding site for AOM1 was identified as SVVYGLRSKS sequence which is immediately adjacent to the RGD motif and also spans the thrombin cleavage site of the human OPN. AOM1 efficiently inhibited OPNa binding to recombinant integrin αvβ3 with an IC50 of 65 nM. Due to its unique binding site, AOM1 is capable of inhibiting OPN cleavage by thrombin which has been shown to produce an OPN fragment that is biologically more active than the full length OPN. Screening of human cell lines identified tumor cells with increased expression of OPN receptors (αvβ3 and CD44v6) such as mesothelioma, hepatocellular carcinoma, breast, and non-small cell lung adenocarcinoma (NSCLC). CD44v6 and αvβ3 were also found to be highly enriched in the monocyte, but not lymphocyte, subset of human peripheral blood mononuclear cells (hPBMCs). In vitro, OPNa induced migration of both tumor and hPBMCs in a transwell migration assay. AOM1 significantly blocked cell migration further validating its specificity for the ligand. OPN was found to be enriched in mouse plasma in a number of pre-clinical tumor model of non-small cell lung cancers. To assess the role of OPN in tumor growth and metastasis and to evaluate a potential therapeutic indication for AOM1, we employed a Kras(G12D-LSL)p53(fl/fl) subcutaneously implanted in vivo model of NSCLC which possesses a high capacity to metastasize into the lung. Our data indicated that treatment of tumor bearing mice with AOM1 as a single agent or in combination with Carboplatin significantly inhibited growth of large metastatic tumors in the lung further supporting a role for OPN in tumor metastasis and progression.
    Full-text · Article · Mar 2012 · Journal of Experimental & Clinical Cancer Research
  • Source
    • "Additionally, osteopontin-deficient vascular smooth muscle cells show increased apoptosis and decreased adherence. This not only provides further evidence for the role of osteopontin in cellular adherence but also in the inhibition of apoptosis (Weintraub et al, 2000). The mechanism by which osteopontin inhibits apoptosis is thought to be via multiple ligand receptor interactions in response to various proapoptotic signals (Denhardt et al, 2001). "
    [Show abstract] [Hide abstract]
    ABSTRACT: A metastatic phenotype can be induced in benign rat mammary cells (Rama 37 cells) by transfecting them with metastasis-inducing DNAs (Met-DNAs). Stable transfection of Met-DNAs increases the level of the metastasis-associated protein, osteopontin. Randomly picked clonal cell lines have been established from the pool of Rama 37 cells transfected with one metastasis-inducing DNA, C9-Met-DNA. In these cell lines, moderate correlation is observed between the copy number of C9-Met-DNA and their metastatic potential (linear regression coefficient, R(2)=0.48). A very close correlation is observed between the cell lines' metastatic potential in vivo and the osteopontin mRNA levels in vitro (R(2)=0.74), but not with another metastasis-associated protein in this system, S100A4 (R(2)=0.21). A close correlation is also observed between osteopontin mRNA levels and the adhesive potential (R(2)=0.91) of the cells, but not with their growth rate in vitro (R(2)=0.03). These observations support the previous suggestion that osteopontin is the direct effector of C9-Met-DNA and that the presence of C9-Met-DNA is necessary, if not sufficient, for the induction of metastasis in vivo in this system. Additionally, these results suggest that Rama 37 cells with increased osteopontin mRNA levels become metastatic not through an increased growth rate, but through an increase in cellular adhesiveness.
    Full-text · Article · Jun 2004 · British Journal of Cancer
Show more