Optimization of iron oxide nanoparticle detection using ultrashort echo time pulse sequences: comparison of T1, T2*, and synergistic T1- T2* contrast mechanisms.

Iron oxide nanoparticles (IONPs) are used in various MRI applications as negative contrast agents. A major challenge is to distinguish regions of signal void due to IONPs from those due to low signal tissues or susceptibility artifacts. To overcome this limitation, several positive contrast strategies have been proposed. Relying on IONP T(1) shortening effects to generate positive contrast is a particularly appealing strategy because it should provide additional specificity when associated with the usual negative contrast from effective transverse relaxation time (T(2)*) effects. In this article, ultrashort echo time imaging is shown to be a powerful technique which can take full advantage of both contrast mechanisms. Methods of comparing T(1) and T(2)* contrast efficiency are described and general rules that allow optimizing IONP detection sensitivity are derived. Contrary to conventional wisdom, optimizing T(1) contrast is often a good strategy for imaging IONPs. Under certain conditions, subtraction of a later echo signal from the ultrashort echo time signal not only improves IONP specificity by providing long T(2)* background suppression but also increases detection sensitivity, as it enables a synergistic combination of usually antagonist T(1) and T(2)* contrasts. In vitro experiments support our theory, and a molecular imaging application is demonstrated using tumor-targeted IONPs in vivo.

Integrins and anoikis.

The loss of integrin-mediated cell-matrix contact induces apoptosis ('anoikis') in certain cell types. Recently it has been shown that protein kinase signaling pathways control anoikis both positively and negatively. Focal adhesion kinase, when activated by integrins, can suppress anoikis. Phosphatidylinositol 3-kinase and the AKT oncoprotein may mediate the anoikis-suppressing effects of focal adhesion kinase. Conversely, the stress-activated protein kinase/Jun amino-terminal kinase pathway promotes anoikis. Latest results indicate that caspase-mediated cleavage of the first component of this latter pathway, MEKK-1, may trigger activation of this pathway in anoikis. In addition, certain integrins may regulate bcl-2 expression levels, possibly adjusting the threshold for anoikis.

A polymeric form of fibronectin has antimetastatic effects against multiple tumor types.

Metastasis accounts for most deaths in cancer patients. Tumor cell adhesion to the extracellular matrix through integrins is thought to be a critical step in metastasis and a potential target for therapeutic intervention. We show here that treatment of human osteosarcoma, melanoma and carcinoma cells with a polymeric form of fibronectin (sFN), before inoculation into nude mice, prevented tumor formation. Intraperitoneally administered sFN significantly reduced lung colonization from intravenously injected tumor cells (experimental metastasis) and from subcutaneous tumors in nude mice (spontaneous metastasis). Treatment with sFN blocked cell spreading and migration in vitro suggesting a possible mechanism for the antimetastatic effect.

Anti-cancer activity of targeted pro-apoptotic peptides.

We have designed short peptides composed of two functional domains, one a tumor blood vessel 'homing' motif and the other a programmed cell death-inducing sequence, and synthesized them by simple peptide chemistry. The 'homing' domain was designed to guide the peptide to targeted cells and allow its internalization. The pro-apoptotic domain was designed to be nontoxic outside cells, but toxic when internalized into targeted cells by the disruption of mitochondrial membranes. Although our prototypes contain only 21 and 26 residues, they were selectively toxic to angiogenic endothelial cells and showed anti-cancer activity in mice. This approach may yield new therapeutic agents.

Fibroblast surface antigen produced but not retained by virus-transformed human cells.

Normal human fibroblasts contain a cell type-specific glycoprotein antigen (SF) that is known to be slowly shed into the medium and to be present also in human serum. Immunofluorescence with anti-SF antibodies showed that SF antigen has a highly nonrandom fibrillar distribution in surface of normal fibroblasts. Simian virus 40-transformed fibroblasts also produced the SF antigen, as shown by radioimmunoassay or immunodiffusion tests, but it was not retained by the surface of these cells. This creates a major difference between the surfaces of normal and malignant cells.

Interaction of soluble fibroblast surface antigen with fribrinogen and fibrin.

A cell-type specific glycoprotein antigen (SFA) from fibroblast surface appears in human plasma and serum. The amount of SFA in serum was reduced if the blood coagulation clot was removed at a low temperature. SFA could be bound to Sepharose-conjugated fibrinogen and to fibrin powder at 0 degrees C and was subsequently released when the temperature was elevated to plus 37 degrees C. This procedure resulted in a 10-fold enrichment of SFA relative to other serum proteins. SFA was found to be concentrated in the cryoprecipitate fraction of human plasma and was copurified with the cold insoluble globulin (CIG) with procedures published for the purification of the latter component. SFA/CIG is not soluble at low temperatures as such and its appearance in the cryoprecipitate fraction of plasma is likely to be due to its affinity to cryofibrinogen evident from these experiments. The biological significance of the interaction of fibroblast surface SFA moleculres with fibrin(ogen) is not known.

Affinity of fibronectin to collagens of different genetic types and to fibrinogen.

Fibronectin, a fibroblast surface protein, was purified from human and chicken plasma and extracts of cultured chicken fibroblasts with affinity chromatography on gelatin coupled to Sepharose particles. A fibronectin-like protein was also isolated from the plasma of Torpedo fish. The collagen binding properties of fibronectin were studied with several genetically distinct collagens. Heat denatured types I, II, and III collagens were equal in their binding capacity and more active than the native collagens or A and B chains. Native type III collagen was more active than the other native collagens. Human and chicken fibronectins showed approximately the same pattern of specificity. Identical specificities were shown by the plasma and fibroblast forms of chicken fibronectin. Two cyanogen bromide peptides of the collagen alpha1 (II) chain, CB8 and CB12, derived from different parts of the chain, were active in fibronectin binding. A polymer of the tripeptide pro-gly-pro, and polyproline were inactive. Fibronectin also binds to fibrinogen and fibrin. Comparison of this binding to collagen binding showed that fibrinogen inhibited binding of fibronectin to collagen, but was less active than native collagen. Two other fibrous proteins, tropoelastin and keratin, did not bind fibronectin. The binding of fibronectin to fibrinogen was inhibited by collagen and incorporation of fibronectin into blood clot in the cold was inhibited by gelatin. These results suggest that the binding of fibronectin to collagen and fibrinogen depends on the same binding site in the fibronectin molecule. It is proposed that cell surface fibronectin mediates attachment of cells to the collagenous extracellular matrix and to a temporary fibrin matrix in a wound.

Distribution of fibroblast surface antigen in the developing chick embryo.

Fibroblast surface (SE) antigen is present in fibrillar surface structures of cultured normal fibroblasts, shed to the extracellular medium, and is also found in circulation (serum and plasma). Malignant fibroblasts (transformed by viruses) do not express SF antigen on the cell surface. In this study the in vivo differentiation and distribution of SF antigen has been investigated in the developing chick embryo using cryostat sections and immunofluorescence. The major findings were: (a) SF antigen was detectable in the loose connective tissue of very early (2-to 3-day old) embryos. (b) Condensation of SF antigen was seen in various boundary membranes such as the glomerular and tubular basement membranes of the kidney, the boundary membranes of the notochord, yolk sac, and vitelline membranes and liver sinusoids. (c) SF antigen was found to be cell-type specific. It was seen as a fibrillar network in the loose connective tissue of different organs but not in the parenchymal cells. It was not found in muscle cells at any stage of development. (d) The antigen was present in the undifferentiated mesenchymal cells of the kidney; but not found after their development into epithelial cells of the secretory tubules. (e) Both in vivo and in fibroblast cultures SF antigen was distributed as a fibrillar network. These data indicate that SF antigen is a "differentiation antigen" restricted to certain cells of mesenchymal origin and character, and that is accumulates in the connective tissue during embryogenesis.

Distribution of fibroblast surface antigen: association with fibrillar structures of normal cells and loss upon viral transformation.

The localization of a cell type-specific, soluble fibroblast surface antigen (SFA) was studied by immunofluorescence and by scanning electron microscopy of the same cells. The antigen had an uneven distribution forming streaks on chick embryo fibroblasts. It was localized to membrane processes and ridges, with a diameter of 50-200 nm. The processes extended from the periphery of the cells to the substratum or to other cells. Trypsin treatment completely removed detectable amounts of SFA. The antigen was detectable within 1 h after trypsin-treated cells were reseeded. The reappearance of SFA correlated with the restoration of membrane processes. Fibroblasts transformed by Rous sarcoma virus (RSV) showed loss of all or most SFA. When normal cells were transformed without subcultivation and trypsinization a fibrillar extracellular network of SFA remained under the transformed fibroblasts while the cells themselves were negative in immunofluorescence. When fibroblasts infected by RSV mutants were transferred to nonpermissive temperature for transformation new SFA was detected within 2 h. These data lead us to propose that loss of stabilizing and anchoring effect of SFA molecules in fibrillar cell surface structures may be critical in altered growth control and malignant transformation.

Mitogenic effect by lipopolysaccharide and pokeweed lectin on density-inhibited chick embryo fibroblasts.

The B lymphocyte mitogens, bacterial lipopolysaccharide (LPS), pokeweed lectin, and tuberculin, induced proliferation in density-inhibited monolayer cultures of chick embryo fibroblasts. The stimulation was seen both as an early increase in sugar uptake and cell volume and later as an increase in thymidine incorporation and cell number. The concentration of LPS maximally stimulating fibroblasts was remarkably low, about 0.1-1 ng/ml. LPS preparation with very different sugar chains gave quite similar results indicating that the architecture of the hydrophilic carbohydrate part is not critical for the mitogenic effect.

Genetic control of alpha-fetoprotein synthesis in the mouse.

To approach the genetic mechanism that turns off the synthesis of alpha-fetoprotein (AFP) after birth, we assumed that a change in this mechanism might affect the low basal level of AFP that can be detected in the adult organism. The concentration of AFP was therefore determined for serum from adult mice of 27 different inbred strains. With one exception, this basal level was between 34 and 173 ng/ml, which is about 10(5)-fold less than the serum concentration at birth. In one strain, BALB/c/J, the AFP level was found to be considerably increased; it was about 10-fold higher than in other strains at 9-10 wk of age. Two other substrains of BALF/c mice showed normally low AFP levels. Kinetic studies show that the rate with which AFP disappears from serum after birth is reduced in BALB/c/J mice as compared to other strains. The increased AFP level of BALB/c/J mice appears to be due to an increased rate of synthesis of AFP, since the rate of catabolism of AFP was found to be normal in these mice. Genetic analysis was performed by crossing BALB/c/J mice with mice having an ordinary AFP level, followed by determination of AFP levels in mice of the F1 and F2 generations as well as in back-cross mice. The results clearly indicate that the increased AFP level in BALB/c/J mice is controlled by a single recessive Mendelian gene, which has been named Raf (for regulation of alphafetoprotein). The Raf gene could be directly involved in the regulation of AFP synthesis, but it may also control AFP levels only indirectly, e.g., by regulating the synthesis of a hormone that controls AFP synthesis.

A common cell-type specific surface antigen in cultured human glial cells and fibroblasts: loss in malignant cells.

Fibroblast surface antigen (SFA) is a high molecular weight protein antigen, first shown on the surface of cultured fibroblasts in fibrillar structures. It is shed to the extracellular medium and also present in the circulation (serum and plasma). Fibroblasts transformed by tumor viruses produce SFA but do not retain it on cell surface. In this report we show that SFA is also present in cultured nonestablished astroglial cells. The glial and fibroblast SFAs are immunologically indistinguishable. Glial cells (three different nonestablished lines) contain more SFA per milligram cellular protein than fibroblasts. SFA was located on cell surface in fibrillar striae that frequently extended out from the cell body. Fluorescence was also found intracellularly in the cytoplasm. Malignant gliomas (astrocytomas) established to grow in culture from human tumor material produced SFA into the growth medium but had very little (lines U-105 MG and U-343 MG) or no detectable (lines U-118 MG, U-251 MG, and U-343 MG-a) cell surface SFA. In cultures of the glioma cells many cells, in particular those that appeared to be in the telophase stage, stained strongly positive for intracellular cytoplasmic SFA. These data demonstrate that similar to fibroblasts transformed experimentally by oncogenic viruses, cells grown from naturally occurring human tumors (glioblastomas) produce SFA but lose ability to retain it on cell surface.

beta1 integrins are critically involved in neutrophil locomotion in extravascular tissue In vivo.

Recruitment of leukocytes from blood to tissue in inflammation requires the function of specific cell surface adhesion molecules. The objective of this study was to identify adhesion molecules that are involved in polymorphonuclear leukocyte (PMN) locomotion in extravascular tissue in vivo. Extravasation and interstitial tissue migration of PMNs was induced in the rat mesentery by chemotactic stimulation with platelet-activating factor (PAF; 10(-7) M). Intravital time-lapse videomicroscopy was used to analyze migration velocity of the activated PMNs, and the modulatory influence on locomotion of locally administered antibodies or peptides recognizing various integrin molecules was examined. Immunofluorescence flow cytometry revealed increased expression of alpha4, beta1, and beta2 integrins on extravasated PMNs compared with blood PMNs. Median migration velocity in response to PAF stimulation was 15.5 +/- 4.5 micron/min (mean +/- SD). Marked reduction (67 +/- 7%) in motility was observed after treatment with mAb blocking beta1 integrin function (VLA integrins), whereas there was little, although significant, reduction (22 +/- 13%) with beta2 integrin mAb. Antibodies or integrin-binding peptides recognizing alpha4beta1, alpha5beta1, or alphavbeta3 were ineffective in modulating migration velocity. Our data demonstrate that cell surface expression of beta1 integrins, although limited on blood PMNs, is induced in extravasated PMNs, and that members of the beta1 integrin family other than alpha4beta1 and alpha5beta1 are critically involved in the chemokinetic movement of PMNs in rat extravascular tissue in vivo.

Antigenic differences in nuclear proteins of normal liver and hepatoma. Identification of a nuclear protein present in hepatocytes but absent in hepatoma cells.

A nuclear antigen was detected in the mouse liver nonhistone protein fraction by using antibodies to whole liver cells. The antigen was purified to homogeneity from perchloric acid extracts of liver tissue. It gave a single band corresponding to tool wt 21,000 in sodium dodecyl sulfate gel electrophoresis. Amino acid and carbohydrate analysis showed predominance of the acidic amino acids, lack of proline, and absence of carbohydrate. Immunofluorescence staining of liver sections confirmed the nuclear localization of the antigen. Its tissue distribution was studied by using radioimmunoassay. Of the various tissues extracted for analysis, the liver contained the highest amounts of the antigen, about 1 mug/mg of solubilized liver protein. Other tissues examined showed 2-4 percent of the amount of antigen present in the liver. Two transplantable hepatomas in C3H/HeJ and C57L/J mice, respectively, and three spontaneous C3H hepatomas showed greatly decreased levels of the antigen compared to normal liver. The amount of antigen in hepatomas varied from nondetectable to 2 percent of the amount of antigen found in the livers of the mice. The antigen was also found in the blood. The antigen was found in high concentrations (up to 13 mg/ml) in the urine of normal mice. This suggests identity with the previously known mouse urinary protein (MUP). In addition to the extremely high urinary output, the properties found to be shared by MUP and the nuclear antigen included similar serum concentrations (2-60 mug/ml), a sex difference with lower values in females, same molecular size as determined by gel filtration, and immunological identity. The nuclear localization of MUP and its disappearance from hepatomas suggest that it may have an important regulatory function.

Tenascin mediates cell attachment through an RGD-dependent receptor.

Tenascin is an extracellular matrix glycoprotein expressed in association with mesenchymal-epithelial interactions during development and in the neovasculature and stroma of undifferentiated tumors. This selective expression of tenascin indicates a specific role in cell matrix interactions. We now show that tenascin can support the adhesion of a variety of cell types, including various human tumor cells, normal fibroblasts, and endothelial cells, all of which can attach to a substrate coated with tenascin. Detailed studies on the mechanism of the tenascin-promoted cell attachment were carried out with the human glioma cell line U251MG. The attachment of these cells and others to tenascin were inhibited specifically by peptides containing the RGD cell attachment signal. Affinity chromatography procedures similar to those that have been used to isolate other adhesion receptors yielded a heterodimeric cell surface protein which bound to a tenascin affinity matrix in an RGD-dependent fashion. One of the subunits of this putative tenascin receptor comigrates with the beta subunit of the fibronectin receptor in SDS-PAGE and cross reacts with antibodies prepared against the fibronectin receptor in immunoblotting. These results identify the tenascin receptor as a member of the fibronectin receptor family within the integrin superfamily of receptors. The cell attachment response on tenascin is distinctly different from that seen on fibronectin, suggesting that cell adhesion and motility may be modulated at those sites where tenascin is expressed in the extracellular matrix.

A fibronectin self-assembly site involved in fibronectin matrix assembly: reconstruction in a synthetic peptide.

The active form of fibronectin is its extracellular matrix form, which allows for the attachment of cells and influences both the growth and migration of cells. The matrix form is assembled by cells; however, many cells are defective in this regard. Several regions within fibronectin have been shown to play a role in matrix assembly by cells. One such region has been localized into the first type III repeat of fibronectin (Chernousov, M. A., F. J. Fogerty, V. E. Koteliansky, and D. F. Mosher. J. Biol. Chem. 266:10851-10858). We have identified this site as a fibronectin-fibronectin binding site and reproduced it as a synthetic peptide. This site is contained in a 14-kD fragment that corresponds to portions of the first two type III repeats. The 14-kD fragment was found to bind to cell monolayers and to inhibit fibronectin matrix assembly. The 14-kD fragment only slightly reduced the binding of fibronectin to cell surfaces but it significantly inhibited the subsequent incorporation of fibronectin into the extracellular matrix. The 14-kD fragment also bound to purified fibronectin and inhibited fibronectin-fibronectin binding. A synthetic 31-amino acid peptide (P1) representing a segment of the 14-kD fragment retained the ability to inhibit fibronectin-fibronectin binding. Peptide P1 specifically bound fibronectin from plasma in affinity chromatography, whereas a column containing another peptide from the 14-kD fragment did not. These results define a fibronectin-fibronectin binding site that appears to promote matrix assembly by allowing the assembly of fibronectin molecules into nascent fibrils. The 14-kD fragment and the P1 peptide that contain this site inhibit matrix assembly by competing for the fibronectin-fibronectin binding.

Distribution of fetal bovine serum fibronectin and endogenous rat cell fibronectin in extracellular matrix.

Normal rat kidney cells were cultured in medium supplemented with normal fetal bovine serum (FBS) or FBS depleted of fibronectin. The cell surface fibronectin of these cultures was visualized by indirect immunofluorescence using species-specific antisera for either rat fibronectin or bovine fibronectin. Anti-rat-fibronectin revealed fibrillar structures on the cells grown in either normal medium or fibronectin-depleted medium. Anti-bovine fibronectin revealed similar fibrillar networks, but only on the cells grown in medium containing bovine fibronectin. Staining in each case was abolished by absorption with the homologous antigen. It appears that exogenous fibronectin was incorporated into the same structures as endogenous fibronectin. This finding suggests that circulating fibronectin may serve as a building block for the assembly of extracellular matrix, possibly by cells which are incapable of synthesizing it.

A cell surface receptor complex for collagen type I recognizes the Arg-Gly-Asp sequence.

To isolate collagen-binding cell surface proteins, detergent extracts of surface-iodinated MG-63 human osteosarcoma cells were chromatographed on affinity matrices of either type I collagen-Sepharose or Sepharose carrying a collagen-like triple-helical peptide. The peptide was designed to be triple helical and to contain the sequence Arg-Gly-Asp, which has been implicated as the cell attachment site of fibronectin, vitronectin, fibrinogen, and von Willebrand factor, and is also present in type I collagen. Three radioactive polypeptides having apparent molecular masses of 250 kD, 70 kD, and 30 kD were distinguishable in that they showed affinity toward the collagen and collagen-like peptide affinity columns, and could be specifically eluted from these columns with a solution of an Arg-Gly-Asp-containing peptide, Gly-Arg-Gly-Asp-Thr-Pro. These collagen-binding polypeptides associated with phosphatidylcholine liposomes, and the resulting liposomes bound specifically to type I collagen or the collagen-like peptide but not to fibronectin or vitronectin or heat-denatured collagen. The binding of these liposomes to type I collagen could be inhibited with the peptide Gly-Arg-Gly-Asp-Thr-Pro and with EDTA, but not with a variant peptide Gly-Arg-Gly-Glu-Ser-Pro. We conclude from these data that these three polypeptides are membrane molecules that behave as a cell surface receptor (or receptor complex) for type I collagen by interacting with it through the Arg-Gly-Asp tripeptide adhesion signal. The lack of binding to denatured collagen suggests that the conformation of the Arg-Gly-Asp sequence is important in the recognition of collagen by the receptor complex.

A peptide isolated from phage display libraries is a structural and functional mimic of an RGD-binding site on integrins.

Many integrins recognize short RGD-containing amino acid sequences and such peptide sequences can be identified from phage libraries by panning with an integrin. Here, in a reverse strategy, we have used such libraries to isolate minimal receptor sequences that bind to fibronectin and RGD-containing fibronectin fragments in affinity panning. A predominant cyclic motif, *CWDDG/LWLC*, was obtained (the asterisks denote a potential disulfide bond). Studies using the purified phage and the corresponding synthetic cyclic peptides showed that *CWDDGWLC*-expressing phage binds specifically to fibronectin and to fibronectin fragments containing the RGD sequence. The binding did not require divalent cations and was inhibited by both RGD and *CWDDGWLC*-containing synthetic peptides. Conversely, RGD-expressing phage attached specifically to immobilized *CWDDGWLC*-peptide and the binding could be blocked by the respective synthetic peptides in solution. Moreover, fibronectin bound to a *CWDDGWLC*-peptide affinity column, and could be eluted with an RGD-containing peptide. The *CWDDGWLC*-peptide inhibited RGD-dependent cell attachment to fibronectin and vitronectin, but not to collagen. A region of the beta subunit of RGD-binding integrins that has been previously demonstrated to be involved in ligand binding includes a polypeptide stretch, KDDLW (in beta 3) similar to WDDG/LWL. Synthetic peptides corresponding to this region in beta 3 were found to bind RGD-displaying phage and conversion of its two aspartic residues into alanines greatly reduced the RGD binding. Polyclonal antibodies raised against the *CWDDGWLC*-peptide recognized beta 1 and beta 3 in immunoblots. These data indicate that the *CWDDGWLC*-peptide is a functional mimic of ligand binding sites of RGD-directed integrins, and that the structurally similar site in the integrin beta subunit is a binding site for RGD.

Isolation of a highly specific ligand for the alpha 5 beta 1 integrin from a phage display library.

Our previous studies showed that the alpha 5 beta 1 integrin selects cysteine pair-containing RGD peptides from a phage display library based on a random hexapeptide. We have therefore searched for more selective peptides for this integrin using a larger phage display library, where heptapeptides are flanked by cysteine residues, thus making the inserts potentially cyclic. Most of the phage sequences that bound to alpha 5 beta 1 (69 of 125) contained the RGD motif. Some of the heptapeptides contained an NGR motif. As the NGR sequence occurs in the cell-binding region of the fibronectin molecule, this sequence could contribute to the specific recognition of fibronectin by alpha 5 beta 1. Selection for high affinity peptides for alpha 5 beta 1 surprisingly yielded a sequence RRETAWA that does not bear obvious resemblance to known integrin ligand sequences. The synthetic cyclic peptide GACRRETAWACGA (*CRRETAWAC*) was a potent inhibitor of alpha 5 beta 1-mediated cell attachment to fibronectin. This peptide is nearly specific for the alpha 5 beta 1 integrin, because much higher concentrations were needed to inhibit the alpha v beta 1 integrin, and there was no effect on alpha v beta 3- and alpha v beta 5-mediated cell attachment to vitronectin. The peptide also did not bind to the alpha IIb beta 3 integrin. *CRRETAWAC* appears to interact with the same or an overlapping binding site in alpha 5 beta 1 as RGD, because cell attachment to *CRRETAWAC* coated on plastic was divalent cation dependent and could be blocked by an RGD-containing peptide. These results reveal a novel binding specificity in the alpha 5 beta 1 integrin.