We have refined a series of in
vitro assays for screening pro- and anti-inflammatory and anti-metastatic
activity of agents being evaluated for commercial development. Our
assays are designed to serve as a set of critical secondary screening
tests to evaluate survivors of primary high throughput screens,
rather than directly advancing to labor-intensive, costly, or otherwise
impractical in vivo assays. Our assays have the following advantages:
a) a high degree of in vivo relevance is achieved by employing a
complete stromal extracellular matrix as a substrate rather than
a synthetic molecule or even isolated connective tissue components;
b) human cells and their products are used for applicability to
human disease; c) assays are carried out in multiwell plates to
provide sufficient replicates for statistical significance. These
assays were initially based on a metabolically labeled stromal extracellular
matrix (ECM) from R22 rat heart smooth muscle cells. Degradation
of the individual ECM components by human neutrophils, macrophages,
or tumor cells can be quantitated by measuring release of radiolabels
incorporated into collagen, noncollagenous proteins, proteoglycans,
or adhesion glycoproteins such as fibronectin. Using this system,
we have successfully screened compounds as inhibitors of human neutrophil
elastase and human matrix metalloproteinases for use in such applications
as cystic fibrosis, acute lung injury, adult respiratory distress
syndrome, and periodontal disease. More recently, we have developed
a similar interstitial ECM from normal human stromal cells to evaluate
inhibitors of serine proteinases and matrix metalloproteinases released
by inflammatory cells and prostate, colon, and breast tumor cell
lines. A goal of this project is to employ assays for determination
of synthetic activity of matrix forming cells for evaluation of
compounds for wound healing and tissue repair. Cell-matrix interactions
which modulate formation of inflammatory cytokines, reactive oxygen
species, and programmed cell death can also be modeled in the assays.
A further goal is to optimize the assays for reproducibility and
extended shelf life with modified protocols for potential marketing
as kits for sale to end-users. Such assays could be attractive as
a service to pharmaceutical companies which outsource components
of their product development programs, or as kits to those companies
which maintain their own R&D operations. |
