The function of proteases in the angiogenic process is not limited to proteolysis of the basement membrane and extracellular matrix, which enables growth of the endothelial sprout Proteases also have indirect effects, as they activate other proteases and pro-angiogenic molecules. Proteases can also release angiogenic growth factors that may be sequestered in the extracellular matrix. In addition, proteolytic cleavage can generate molecules that inhibit angiogenesis. Among the serine proteases, the plasmin/ plasminogen activator system, in particular, is thought to play a major role.
Previous work utilised a homology cloning approach on mRNA isolated from endothelial cells undergoing tube formation in a matrigel culture. This exploited the conserved motifs of serine proteases, utilising template mRNA isolated from human dermal microvascular endothelial cells in culture. In this study, amplified DNA was cloned and sequenced. The sequence of about 100 cloned inserts was established. Several serine proteases were detected (acrosin, CTRL-1, neurosin, neurotrypsin and testisin; as well as tPA, a previously-described endothelial cell product) As the expression of a relatively uncharacterised protein that is currentiy classified as a serine protease (PSP) was also detected, the likely properties of this protein were investigated.
Further work by collaborators involved specific-primed RT-PCR on various in-vitro models of angiogenesis, in order to confirm or disprove these results and also to investigate the expression of other candidate genes encoding both secreted and membrane-anchored serine proteases that could be associated with microvascular endothelial tubular networks. Expression of acrosin, neurosin, neurotrypsin, PSP, testisin and tPA was confirmed, and in addition, hepsin, MT-SPl and TMPRSS2 were detected.
This study included in-situ hybridisation (ISH) and immunohistochemistry (IHC) experiments in order to investigate the cellular source of serine protease expression in human tumour tissues. Perivascular signals were obtained using ISH for acrosin, hepsin, MT-SPl, neurosin, neurotrypsin, PSP, testisin and TMPRSS2, although in most instances, the signals were not restricted to the perivascular space. As the antibody had previously been synthesized, anti-testisin IHC was performed and revealed testisin protein expression in blood vessels both with and without angiogenic potential. The finer resolution which IHC afforded enabled the localisation of testisin expression to particular cell types, namely the endothelial cells, pericytes, and smooth muscle cells of blood vessels. These two findings indicate that testisin expression is present in blood vessels, but may not be restricted functionally to the process of angiogenesis.
Finally, in order to investigate the role of certain regulatory cytokines on serine protease expression, endothelial cells were cultured and treated with tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and bacterial endotoxin lipopolysaccharide (LPS). Specific-primed RT-PCR was performed on mRNA isolated from these cells. These stodies provided preliminary results suggesting that, for microvascular endothelial cells grown on plastic, under the experimental conditions used, the expression of tPA and neurotrypsin was downregulated by TNFα and IFNγ; and the expression of acrosin and CTRL-1 was upregulated by LPS.
This study has identified several serine proteases expressed by human dermal microvascular endothelial cells. The results were confirmed in-vivo, and identify several potential targets for anti-angiogenic therapy.