Simon Goodman

Pharmacological Inhibition of Integrin αvβ3 Aggravates Experimental Liver Fibrosis and Suppresses Hepatic Angiogenesis

The vitronectin receptor integrin αvβ3 promotes angiogenesis by mediating migration and proliferation of endothelial cells, but also drives fibrogenic activation of hepatic stellate cells (HSCs) in vitro. Expecting antifibrotic synergism, we studied the effect of αvβ3 inhibition in two in vivo models of liver fibrogenesis. Liver fibrosis was induced in rats by way of bile duct ligation (BDL) for 6 weeks or thioacetamide (TAA) injections for 12 weeks. A specific αvβ3 (αvβ5) inhibitor (Cilengitide) was given intraperitoneally twice daily at 15 mg/kg during BDL or after TAA administration. Liver collagen was determined as hydroxyproline, and gene expression was quantified by way of quantitative polymerase chain reaction. Liver angiogenesis, macrophage infiltration, and hypoxia were assessed by way of CD31, CD68 and hypoxia‐inducible factor‐1α immunostaining. Cilengitide decreased overall vessel formation. This was significant in portal areas of BDL and septal areas of TAA fibrotic rats and was associated with a significant increase of liver collagen by 31% (BDL) and 27% (TAA), and up‐regulation of profibrogenic genes and matrix metalloproteinase‐13. Treatment increased gamma glutamyl transpeptidase in both models, while other serum markers remained unchanged. αvβ3 inhibition resulted in mild liver hypoxia, as evidenced by up‐regulation of hypoxia‐inducible genes. Liver infiltration by macrophages/Kupffer cells was not affected, although increases in tumor necrosis factor α, interleukin‐18, and cyclooxygenase‐2 messenger RNA indicated modest macrophage activation. Conclusion: Specific inhibition of integrin αvβ3 (αvβ5) in vivo decreased angiogenesis but worsened biliary (BDL) and septal (TAA) fibrosis, despite its antifibrogenic effect on HSCs in vitro. Angiogenesis inhibitors should be used with caution in patients with hepatic fibrosis. (HEPATOLOGY 2009.)

Matched rabbit monoclonal antibodies against αv-series integrins reveal a novel αvβ3-LIBS epitope, and permit routine staining of archival paraffin samples of human tumors.

Summary The relationship between integrin expression and function in pathologies is often contentious as comparisons between human pathological expression and expression in cell lines is difficult. In addition, the expression of even integrins &agr;v&bgr;6 and &agr;v&bgr;8 in tumor cell lines is not comprehensively documented. Here, we describe rabbit monoclonal antibodies (RabMabs) against the extracellular domains of &agr;v integrins that react with both native integrins and formalin fixed, paraffin embedded (FFPE) human tissues. These RabMabs, against &agr;v&bgr;3 (EM22703), &agr;v&bgr;5 (EM09902), &agr;v&bgr;6 (EM05201), &agr;v&bgr;8 (EM13309), and pan-&agr;v (EM01309), recognize individual integrin chains in Western blots and in flow cytometry. EM22703 detected a ligand-induced binding site (LIBS), reporting an epitope enhanced by the binding of an RGD-peptide to &agr;v&bgr;3. &agr;v&bgr;8 was rarely expressed in human tumor specimens, and weakly expressed in non-small-cell lung carcinoma (NSCLC). However, ovarian carcinoma cell lines expressed &agr;v&bgr;8, as did some melanoma cells, whereas U87MG glioma lacked &agr;v&bgr;8 expression. We observed an unexpected strong expression of &agr;v&bgr;6 in tumor samples of invasive ductal breast adenoma, colorectal carcinoma (CRC), and NSCLC. &agr;v&bgr;3 was strongly expressed in some invasive NSCLC cohorts. Interestingly, PC3 prostate cell and human prostate tumors did not express &agr;v&bgr;3. The RabMabs stained plasma membranes in FFPE-immunohistochemistry (IHC) samples of tumor cell lines from lung, ovary, colon, prostate, squamous cell carcinoma of head and neck (SCCHN), breast, and pancreas carcinomas. The RabMabs are unique tools for probing &agr;v integrin biology, and suggest that especially &agr;v&bgr;6 and &agr;v&bgr;8 biologies still have much to reveal.

The path to VICTORy – a beginner's guide to success using commercial research antibodies

ABSTRACT Commercial research antibodies are crucial tools in modern cell biology and biochemistry. In the USA some

Cyclic RGD peptides with a novel mimetic

2 billion a year are spent on them, but many are apparently not fit-for-purpose, and this may contribute to the ‘reproducibility crisis’ in biological sciences. Inadequate antibody validation and characterization, lack of user awareness, and occasional incompetence amongst suppliers have had immense scientific and personal costs. In this Opinion, I suggest some paths to make the use of these vital tools more successful. I have attempted to summarize and extend expert views from the literature to suggest that sustained routine efforts should made in: (1) the validation of antibodies, (2) their identification, (3) communication and controls, (4) the training of potential users, (5) the transparency of original equipment manufacturer (OEM) marketing agreements, and (5) in a more widespread use of recombinant antibodies (together denoted the ‘VICTOR’ approach). Summary: Much anguish, talent, time and money are expended on cell biology using inadequately characterized commercial research antibodies, and this can lead to bad science. I summarize the situation and suggest accessible solutions.

Anti-.alpha.v-integrin monoclonal antibody

The integrins and their ligands regulate a range of cell-cell and cell-matrix interactions. The αv β 3 integrin is expressed on tumor-induced angiogenic blood vessels which makes inhibitors of this receptor valuable tools for therapeutic purposes [1]. A number of cyclic peptides containing the Arg–Gly–Asp(RGD) sequence were reported to be selective inhibitors of this integrin [2]. cyclo(–RGD D -Phe–Val–) [c(RGDfV)], for example, serves as a standard in many biological assays. Whereas the RGD part of the molecule is very sensitive to changes, the remaining two amino acids, D-Phe and Val, were exchanged by a variety of amino acids and building blocks without loosing affinity or selectivity for the αv β3 integrin [3,4]. We have synthesized a substituted 2-(3-aminomethyl-phenylamino)-propionic acid building block and incorporated it into cyclic RGD peptides. The 3-aminomethyl-phenylamine (AMP) residue is similar to the Mamb (m-(aminomethyl)benzoic acid) residue described by A.C. Bach, II, and co-workers [5].