Project Details

Description

We have demonstrated recently that Macrophage-derived angiogenic
activity (MDAA) is either identical or closely related
immunologically to Tumor Necrosis Factor-alpha (TNF-alpha).
Recombinant (r) TNF-alpha is potently angiogenic in vivo in the rat
cornea and chick chorioallantoic membrane, is a chemoattractant
for bovine capillary endothelial cells (BCE's) in vitro, and
induces confluent monolayers of BCE's cultured on collagen gels to
invade the gels and from capillary-like tubular structures. MDAA
in conditioned media of macrophage cultures is neutralized by a
rabbit anti-murine TNF-alpha polyclonal antibody. We have also
shown that Transforming Growth Factor-beta (TGF-beta), a product
of both normal (platelets and activated lymphocytes) as well as
transformed cells, is a potent chemoattractant for human monocytes
in vitro, with maximal activity in the femtomolar. At picomolar
concentrations, TGF-beta induces expression of angiogenic activity
by monocytes. The projects in this application are designed to
study: (a) Events involved in the activation of
monocyte/macrophage expression of angiogenic activity (MDAA/TNF-
alpha). TGF-beta and oxygen/lactate concentrations will be
studied. C-DNA clones obtained from Genentech, Inc. will be used
to probe for TNF-alpha, TGF-alpha and TGF-beta mRNA levels in
monocytes/macrophages. Levels of expressed protein will also be
determined. (b) Structural features of TNF-alpha that relate to
its angiogenic activity. Five monoclonal antibodies to TNF-alpha,
two that neutralize cytotoxic activity, three that do not, will b
compared for anti-angiogenic activity. Specific proteolytic and
chemical cleavage of TNF-alpha will be carried out, and the
peptides tested for angiogenic and cytotoxic activity. Synthetic
peptide analogs of active sequences will ultimately be examined in
relation to potential inhibitors. (c) The mechanism of action of
TNF-alpha as an angiogenic agent, by studying its effects on
capillary endothelial cells in culture. Endothelial cell receptors
for TNF-alpha will be studied using 125I-labelled TNF-alpha. The
effects of TNF-alpha on basic Fibroblast Growth Factor (bFGF) mRNA
levels will be examined. Secretion of metalloproteinases, such as
collagenase, and TIMP (Tissue Inhibitor of Metalloproteinases) will
be studied. (d) The expression of TNF-alpha in vivo will be
examined in wounds, inflammation and tumors, by in situ
hybridization. Either a labelled oligonucleotide probe for TNF-
alpha, or an RNA probe ("Riboprobe"), will be used. (e) The
effects of TNF-alpha on wound repair in vivo will be studied, using
the implanted polyvinyl (lvalon) sponge and the implanted Gortex
micropermeable tubing models.
StatusFinished
Effective start/end date1/1/816/30/95

Funding

  • National Institutes of Health
  • National Institutes of Health: $159,229.00
  • National Institutes of Health: $130,487.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health

ASJC

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)

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