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D B Rifkin

Publications and source records attributed to D B Rifkin.

At least 19 recordsLinked to original sources
PubMed1999

The integrin alpha v beta 6 binds and activates latent TGF beta 1: a mechanism for regulating pulmonary inflammation and fibrosis.

Transforming growth factor beta (TGF beta) family members are secreted in inactive complexes with a latency-associated peptide (LAP), a protein derived from the N-terminal region of the TGF beta gene product. Extracellular activation of these complexes is a critical but incompletely understood step in regulation of TGF beta function in vivo. We show that TGF beta 1 LAP is a ligand for the integrin alpha v beta 6 and that alpha v beta 6-expressing cells induce spatially restricted activation of TGF beta 1. This finding explains why mice lacking this integrin develop exaggerated inflammation and, as we show, are protected from pulmonary fibrosis. These data identify a novel mechanism for locally regulating TGF beta 1 function in vivo by regulating expression of the alpha v beta 6 integrin.

3T3 Cells↗
PubMed1999

Diltiazem reduces retinal neovascularization in a mouse model of oxygen induced retinopathy.

PURPOSE: To assess the effect of diltiazem, a calcium channel blocking agent, on oxygen induced retinopathy (OIR) in a mouse model using neovascular nuclei quantitation and a quantitative scoring system based on examining fluorescein perfused retinal whole mount preparations. METHODS: The mouse model of oxygen induced retinopathy consisting of a 5 day exposure to 75% oxygen from postnatal day 7 to 12 was used to produce retinal neovascularization. Fluorescein conjugated dextran angiography of retinal vasculature was performed and retinal whole mounts were prepared to score features of retinopathy. The parameters that were scored in a masked fashion included blood vessel growth, blood vessel tuft formation, extra retinal neovascularization, degree of central vasoconstriction, retinal hemorrhage, and tortuosity of vessels. Diltiazem (0.05-0.5 mg/kg/day subcutaneously for five days) was administered to mice pups during exposure to oxygen to determine if calcium channel blockade altered retinopathy. In addition, quantification of retinal neovascular nuclei was performed in a masked fashion with periodic acid Schiff (PAS) staining of frozen eye sections. RESULTS: Animals that were exposed to hyperoxia for five days had a median (25th, 75th quartile) retinopathy score of 9 (8,11) versus control animals that had a retinopathy score of 1 (0,1) with p<0.001. Subscores for blood vessel growth, blood vessel tufts, extra-retinal neovascularization, central vasoconstriction, hemorrhage, and blood vessel tortuosity were all significantly different between control and treated animals. In addition, quantification of neovascular nuclei showed a significant increase in the number of nuclei extending beyond the inner limiting membrane into the vitreous in hyperoxic treated animals. Diltiazem at doses of 0.2 and 0.5 mg/kg/day improved the retinopathy as measured by the total retinopathy score [5 (4,6) and 4 (3.75,5.25), respectively]. The average number of extraretinal neovascular nuclei per retinal section (mean +/-standard deviation) was significantly decreased by diltiazem at doses of 0.2 and 0.5 mg/kg/day (31.4+/-18.8 and 20.9+/-6.9, respectively) when compared to hyperoxic treated animals (56.1+/-21.5). CONCLUSIONS: Diltiazem reduces oxygen induced retinopathy in the mouse as measured by a scoring system based on a retinal whole mount method of retinal neovascularization and by quantification of extra retinal neovascular nuclei.

Animals↗
PubMed1998

Intracellular association of FGF-2 with the ribosomal protein L6/TAXREB107.

By using the yeast two-hybrid system, we identified the ribosomal protein L6/TAXREB107 as an intracellular partner for FGF-2. L6/TAXREB107 also mediates the DNA binding of the HTLV-1 transactivator Tax. In vitro binding experiments indicated that both the high-molecular-weight forms (HMW) and the 18-kDa form of FGF-2 bind to L6/TAXREB107. Deletion analysis suggested that L6/TAXREB107 has two binding sites for HMW FGF-2 and one binding site for 18-kDa FGF-2, implying that the unique N-terminal extension of the HMW FGF-2 is one of the binding domains for L6/TAXREB107. Transfection assays showed that high expression of either HMW or 18 kDa FGF-2 stimulates Tax-mediated transactivation in NIH 3T3 cells. This result suggests a possible role of FGF-2 in Tax-mediated HTLV-1 transformation as well as FGF-2 binding to ribosomes and/or their precursors.

Animals↗
PubMed1998

Irsogladine maleate inhibits angiogenesis in wild-type and plasminogen activator-deficient mice.

BACKGROUND: The activation of the zymogen plasminogen to the serine protease plasmin by urokinase-type (uPA) and tissue-type (tPA) plasminogen activators (PA) is an important event in a variety of physiologic and pathophysiologic processes in mammals. Enhanced PA activity occurs during angiogenesis and has been correlated in vitro and in vivo with increased tumor aggressiveness and is an indicator of poor prognosis in a variety of tumors in humans. Preliminary studies suggest that the antiulcer drug irsogladine maleate (IM) diminishes PA activity in vitro and may inhibit angiogenesis in vivo. To define the precise mechanism of angiogenesis inhibition by IM in vivo, we tested the ability of IM to blunt angiogenesis in a mouse cornea neovascularization model performed in wild-type and PA-knockout mice. METHODS: Three days prior to pellet implantation, groups of C57Bl/6 wild-type, uPA-deficient (upA-/-), and tPA-deficient (tPA-/-) mice received IM (300 mg/kg), IM (500 mg/kg), or vehicle (0.5% carboxymethylcellulose) via oral gavage. After 3 days of treatment, hydron polymer-coated pellets of sucrose aluminum sulfate containing 100 ng of basic fibroblast growth factor (bFGF) were inserted into surgically created pockets in the cornea of each mouse. On postoperative day 6, the neovascularization of each cornea was evaluated by a blinded observer using slit lamp microscopy and photographed. Angiogenesis was quantified by calculating vascular area (mm2) +/- SEM using a modified formula for a half ellipse that incorporates calibrated vessel measurements [Vessel length (mm) x Clock hours x pi x 0.2]. RESULTS: IM treatment (300 and 500 mg/kg/day) resulted in a dose-dependent reduction of angiogenesis in wild-type mice by 21 and 45.3% (P < 0.02, P < 0.001), in tPA-deficient mice by 42.6 and 46% (P < 0.001, P < 0.001), and in uPA-deficient mice by 27.2 and 46% (P < 0.05, p < 0.001), respectively. No quantitative differences in neovascularization were observed in either treatment group between transgenic mouse strains. No toxicity was noted in any group. CONCLUSION: IM inhibits bFGF-induced angiogenesis in wild-type, tPA-knockout, and uPA-knockout mice. The observation that IM significantly diminishes angiogenesis in both PA-deficient mice and wild-type mice suggests that the mechanism of action of IM may be independent of plasminogen activation.

Animals↗
PubMed1998

Fibroblast growth factor-2 (FGF-2) induces vascular endothelial growth factor (VEGF) expression in the endothelial cells of forming capillaries: an autocrine mechanism contributing to angiogenesis.

FGF-2 and VEGF are potent angiogenesis inducers in vivo and in vitro. Here we show that FGF-2 induces VEGF expression in vascular endothelial cells through autocrine and paracrine mechanisms. Addition of recombinant FGF-2 to cultured endothelial cells or upregulation of endogenous FGF-2 results in increased VEGF expression. Neutralizing monoclonal antibody to VEGF inhibits FGF-2-induced endothelial cell proliferation. Endogenous 18-kD FGF-2 production upregulates VEGF expression through extracellular interaction with cell membrane receptors; high-Mr FGF-2 (22-24-kD) acts via intracellular mechanism(s). During angiogenesis induced by FGF-2 in the mouse cornea, the endothelial cells of forming capillaries express VEGF mRNA and protein. Systemic administration of neutralizing VEGF antibody dramatically reduces FGF-2-induced angiogenesis. Because occasional fibroblasts or other cell types present in the corneal stroma show no significant expression of VEGF mRNA, these findings demonstrate that endothelial cell-derived VEGF is an important autocrine mediator of FGF-2-induced angiogenesis. Thus, angiogenesis in vivo can be modulated by a novel mechanism that involves the autocrine action of vascular endothelial cell-derived FGF-2 and VEGF.

3T3 Cells↗
PubMed1998

Hyperoxia stimulates endothelin-1 secretion from endothelial cells; modulation by captopril and nifedipine.

PURPOSE: Retinopathy of prematurity (ROP) is a vasoproliferative condition that can result in severe visual impairment and blindness in preterm babies. Two conditions seen very early in radioimmunoassay (ROP) are vasoconstriction and vaso-obliteration. A potent vasoconstrictor secreted by endothelial cells is endothelin-1 (ET-1). Premature birth results in a relative systemic hyperoxia, compared to the in utero oxygen milieu. We tested the hypothesis that hyperoxia increases ET-1 expression as a possible mechanism for vasoconstriction in the retinal vasculature. METHODS: Bovine retinal endothelial cells and adrenal capillary endothelial cells were isolated and maintained in culture. Cells were exposed to control or hyperoxic culture conditions for 24 h, with and without addition of captopril and nifedipine. Media was collected and assayed for ET-1 by ROP. In addition, cell counts and secreted LDH assays were performed. RESULTS: Conditioned media from cultured bovine retinal and adrenal endothelial cells exposed to hyperoxic culture conditions for 24 h were found to have higher levels of ET-1 than conditioned media from normoxic control cells. Captopril (10(-6) M and 10(-4) M) and nifedipine (10(-6) M and 10(-4) M) inhibited ET-1 release from hyperoxia-exposed endothelial cells. Under normoxic conditions, ET-1 release was inhibited by 10(-4) M captopril or 10(-4) M nifedipine. CONCLUSIONS: These results demonstrate that (1) hyperoxia stimulates in vitro ET-1 secretion in bovine retinal and adrenal capillary endothelial cells, and (2) captopril and nifedipine downregulate ET-1 secretion under normoxic and hyperoxic culture conditions, in a dose-dependent fashion. We speculate that ET-1 may be involved in retinal vessel vasoconstriction seen early in the development of ROP. Further, ACE inhibitors and calcium-channel blocking agents, such as captopril and nifedipine, may provide an avenue for blocking vasoconstriction in ROP.

Adrenal Glands↗
PubMed1998

Interactions between growth factors and integrins: latent forms of transforming growth factor-beta are ligands for the integrin alphavbeta1.

The multipotential cytokine transforming growth factor-beta (TGF-beta) is secreted in a latent form. Latency results from the noncovalent association of TGF-beta with its processed propeptide dimer, called the latency-associated peptide (LAP); the complex of the two proteins is termed the small latent complex. Disulfide bonding between LAP and latent TGF-beta-binding protein (LTBP) produces the most common form of latent TGF-beta, the large latent complex. The extracellular matrix (ECM) modulates the activity of TGF-beta. LTBP and the LAP propeptides of TGF-beta (isoforms 1 and 3), like many ECM proteins, contain the common integrin-binding sequence RGD. To increase our understanding of latent TGF-beta function in the ECM, we determined whether latent TGF-beta1 interacts with integrins. A549 cells adhered and spread on plastic coated with LAP, small latent complex, and large latent complex but not on LTBP-coated plastic. Adhesion was blocked by an RGD peptide, and cells were unable to attach to a mutant form of recombinant LAP lacking the RGD sequence. Adhesion was also blocked by mAbs to integrin subunits alphav and beta1. We purified LAP-binding integrins from extracts of A549 cells using LAP bound to Sepharose. alphavbeta1 eluted with EDTA. After purification in the presence of Mn2+, a small amount of alphavbeta5 was also detected. A549 cells migrated equally on fibronectin- and LAP-coated surfaces; migration on LAP was alphavbeta1 dependent. These results establish alphavbeta1 as a LAP-beta1 receptor. Interactions between latent TGF-beta and alphavbeta1 may localize latent TGF-beta to the surface of specific cells and may allow the TGF-beta1 gene product to initiate signals by both TGF-beta receptor and integrin pathways.

Cell Adhesion↗
PubMed1998

Structure and activation of the large latent transforming growth factor-Beta complex.

BACKGROUND: Many cytokines regulate processes involved in the pathogenesis of proliferative vitreoretinopathy. Transforming growth factor-beta (TGF-beta) is an example of a pluripotent growth factor that regulates cell proliferation, extracellular matrix (ECM) deposition, cell migration, and differentiation--all biological activities involved in the formation and progression of proliferative vitreoretinopathies. METHODS: A review of experimental results that demonstrate how vascular cells generate biologically active TGF-beta is presented. Most cell types--including endothelial cells and pericytes, which form the retinal microvasculature--express TGF-beta as a large latent TGF-beta complex. Mature TGF-beta, the biologically active form, must be generated from the large latent complex before it can signal by binding to its high affinity cell surface receptors. RESULTS: A critical step in regulating TGF-beta effects may be the activation of the large latent TGF-beta complex. Activation of the complex can be achieved by chemical and enzymatic treatments, or by various cell systems. We have identified that co-culturing bovine smooth muscle cells or pericytes and endothelial cells generates active TGF-beta. CONCLUSION: The mechanism of latent TGF-beta activation self-regulates through effectors of plasmin generation. Studying TGF-beta generation by co-cultures of pericytes and endothelial cells can provide us with insights into how disruption of latent TGF-beta activation may lead to unregulated endothelial proliferation, ECM deposition, and cellular infiltration, as observed clinically in neovascular- and fibrotic-related pathologies.

Animals↗
PubMed1997

Latent transforming growth factor-beta binding protein domains involved in activation and transglutaminase-dependent cross-linking of latent transforming growth factor-beta.

Transforming growth factor-beta (TGF-beta) is secreted by many cell types as part of a large latent complex composed of three subunits: TGF-beta, the TGF-beta propeptide, and the latent TGF-beta binding protein (LTBP). To interact with its cell surface receptors, TGF-beta must be released from the latent complex by disrupting noncovalent interactions between mature TGF-beta and its propeptide. Previously, we identified LTBP-1 and transglutaminase, a cross-linking enzyme, as reactants involved in the formation of TGF-beta. In this study, we demonstrate that LTBP-1 and large latent complex are substrates for transglutaminase. Furthermore, we show that the covalent association between LTBP-1 and the extracellular matrix is transglutaminase dependent, as little LTBP-1 is recovered from matrix digests prepared from cultures treated with transglutaminase inhibitors. Three polyclonal antisera to glutathione S-transferase fusion proteins containing amino, middle, or carboxyl regions of LTBP-1S were used to identify domains of LTBP-1 involved in cross-linking and formation of TGF-beta by transglutaminase. Antibodies to the amino and carboxyl regions of LTBP-1S abrogate TGF-beta generation by vascular cell cocultures or macrophages. However, only antibodies to the amino-terminal region of LTBP-1 block transglutaminase-dependent cross-linking of large latent complex or LTBP-1. To further identify transglutaminase-reactive domains within the amino-terminal region of LTBP-1S, mutants of LTBP-1S with deletions of either the amino-terminal 293 (deltaN293) or 441 (deltaN441) amino acids were expressed transiently in CHO cells. Analysis of the LTBP-1S content in matrices of transfected CHO cultures revealed that deltaN293 LTBP-1S was matrix associated via a transglutaminase-dependent reaction, whereas deltaN441 LTBP-1S was not. This suggests that residues 294-441 are critical to the transglutaminase reactivity of LTBP-1S.

Animals↗
PubMed1997

TGF-beta latency: biological significance and mechanisms of activation.

Transforming growth factor (TGF-) beta is secreted as a latent complex in which the mature growth factor remains associated with its propeptide. In order to elicit a biological response, the cytokine must be released from the latent complex, a process termed latent TGF-beta activation or TGF-beta formation. Although latent TGF-beta activation is a critical step in the regulation of its activity, little is known about the molecular mechanisms that lead to the production of active TGF-beta. In this article, we present an overview of the data available on this topic, and we propose a tentative model for the mechanism of TGF-beta formation based upon the observations with different cell systems and on recent findings on the structure of the latent TGF-beta complex.

Animals↗
PubMed1997

Latent transforming growth factor-beta: structural features and mechanisms of activation.

Transforming growth factor-beta are cytokines with a wide range of biological effects. They play a pathologic role in inflammatory and fibrosing diseases such as nephrosclerosis. TGF-beta s are secreted in a latent form due to noncovalent association with latency associated peptide (LAP), which is a homodimer formed from the propeptide region of TGF-beta. LAP is disulfide linked to another protein, latent TGF-beta binding protein (LTBP). LTBP has features in common with extracellular matrix proteins, and targets latent TGF-beta to the matrix. Activation of latent TGF-beta can be accomplished in vitro by denaturing treatments, plasmin digestion, ionizing radiation and interaction with thrombospondin. The mechanisms by which latent TGF-beta is activated physiologically are not well understood. Results to date suggest an important role for proteases, particularly plasmin, although other mechanisms probably exist. A general model of activation is proposed in which latent TGF-beta is released from the extracellular matrix by proteases, localized to cell surfaces, and activated by cell-associated plasmin.

Animals↗
PubMed1997

Urokinase-type plasminogen activator-deficient mice are predisposed to staphylococcal botryomycosis, pleuritis, and effacement of lymphoid follicles.

Urokinase-type plasminogen activator (uPA) is thought to be an important mediator in the proteolytic degradation of extracellular matrix components observed in a wide variety of normal physiological and pathological conditions. However, the phenotype of a recently developed strain of urokinase-deficient (uPA-/-) mice appears to be normal when maintained under ideal nonstressful conditions. We report an outbreak of botryomycosis, an unusual staphylococcal infection, in a colony of uPA-deficient mice. A detailed histological examination of these uPA-deficient animals also revealed a variety of previously unreported phenotypic abnormalities such as pleuritis and the effacement of lymphoid follicles in the regional lymph nodes and spleen. Additional phenotypic abnormalities such as dystrophic calcifications and rectal prolapse were also observed in the uPA-deficient population. These abnormalities were also noted in ostensibly healthy uPA-deficient animals. Botryomycosis did not affect a colony of wild-type (uPA+/+) animals maintained concurrently under identical conditions in the same room. The peculiar predisposition of the uPA-deficient animals to this rare bacterial infection and the development of phenotypic abnormalities associated with the targeted disruption the uPA gene suggests that uPA contributes significantly to the cutaneous microenvironment and is additional evidence of the extensive involvement of the plasminogen activators in mammalian physiology.

Abscess↗
PubMed1996

Identification and characterization of an eight-cysteine repeat of the latent transforming growth factor-beta binding protein-1 that mediates bonding to the latent transforming growth factor-beta1.

Most cultured cell types secrete small latent transforming growth factor-beta (TGF-beta) as a disulfide-bonded complex with a member of the latent TGF-beta binding protein (LTBP) family. Using the baculovirus expression system, we have mapped the domain of LTBP-1 mediating covalent association with small latent TGF-beta1. Coexpression in Sf9 cells of small latent TGF-beta1 with deletion mutants of LTBP-1 showed that the third eight-cysteine repeat of LTBP-1 is necessary and sufficient for covalent interaction with small latent TGF-beta1. Analysis by mass spectrometry of this eight-cysteine repeat, produced as a recombinant peptide in Sf9 cells, confirmed that it was N-glycosylated, as expected from the primary sequence. No other post-translational modifications of this domain were detected. Alkylation of the recombinant peptide with vinyl pyridine failed to reveal any free cysteines, indicating that, in the absence of small latent TGF-beta, the eight cysteines of this domain are engaged in intramolecular bonds. These data demonstrate that the third LTBP-1 eight-cysteine repeat recognizes and associates covalently with small latent TGF-beta1 through a mechanism that does not require any specific post-translational modification of this domain. They also suggest that this domain adopts different conformations depending on whether it is free or bound to small latent TGF-beta.

Animals↗
PubMed1996

Integrin regulation by endogenous expression of 18-kDa fibroblast growth factor-2.

The three high molecular weight (HMW) forms of fibroblast growth factor-2 (FGF-2) have a distinct intracellular localization and differentially affect cell mobility and growth compared with the fourth 18-kDa form. To characterize further the effects of the 18-kDa and HMW forms of FGF-2, we have examined their ability to modulate integrin expression. Transfected NIH 3T3 cells expressing only 18-kDa FGF-2 exhibited increased cell surface levels of alpha5beta1, whereas cells expressing only HMW FGF-2 exhibited cell surface alpha5beta1 levels similar to parental cells. When cells synthesizing 18-kDa FGF-2 were transfected with a cDNA encoding a dominant negative FGF receptor, alpha5beta1 cell surface levels decreased. Immunoprecipitation of biosynthetically labeled cells indicated that expression of 18-kDa FGF-2 increased the biosynthesis and rate of maturation of alpha5. Northern blot analysis showed that 18-kDa FGF-2 increases the level of the alpha5 subunit mRNA but does not affect beta1 subunit transcript levels. Experiments utilizing luciferase reporter gene activity revealed increased alpha5 promoter activity in cells expressing 18-kDa FGF-2 indicating that the enhanced alpha5 transcript level is due to modulation of the transcription rate. Therefore, interaction of 18-kDa FGF-2 with FGF receptors results in changes in alpha5beta1 biosynthesis and processing. In contrast, endogenous expression of HMW FGF-2 does not mediate this effect.

3T3 Cells↗
PubMed1996

Induction of primary cutaneous melanocytic neoplasms in urokinase-type plasminogen activator (uPA)-deficient and wild-type mice: cellular blue nevi invade but do not progress to malignant melanoma in uPA-deficient animals.

Evidence suggests that the plasminogen activators (PAs), in particular urokinase-type PA (uPA), play a pivotal role in tumor invasion and metastasis. We studied the contribution of the PAs to the malignant phenotype through the chemical induction of melanocytic neoplasms in uPA-deficient mice. Primary tumors were induced and promoted concurrently in 35 uPA-/- deficient and 35 uPA+/+ wild-type mice using a single application of 7,12-dimethylbenz(a)anthracene followed by repetitive applications of croton oil. Animals were sacrificed at 60-day intervals for 1 year. At necropsy, the four largest pigmented lesions in each animal were excised, characterized histologically, and evaluated microscopically for evidence of invasion. The regional lymph nodes, lungs, and solid abdominal visceral organs were sectioned and examined microscopically for evidence of metastatic disease. Cellular blue nevi were induced in 100% of uPA-/- and uPA+/+ promoted animals. Although a reduction in the radial and vertical progression of these lesions was noted in the uPA-deficient mice compared with the wild-type group, more than 95% of cellular blue nevi induced in both groups of animals invaded the underlying tissues. These lesions did not metastasize to the regional lymph nodes. Malignant melanoma arose in 5 of 35 (14.3%) of promoted wild-type mice. These tumors were locally aggressive, produced tissue-type PA, but were not metastatic to the regional nodes, lungs, or abdominal viscera. These results indicate that the invasive capability of melanocytic lesions may depend more on tissue-type PA than uPA activity. No melanomas were induced in the uPA-/- mice. The resistance of the uPA -/- strain to melanoma induction suggests that uPA contributes to malignant progression. We propose that the absence of uPA negatively affects tumorigenesis by decreasing the liberation and availability of growth factors such as basic fibroblast growth factor.

Animals↗