Vedolizumab Antagonizes MAdCAM-1-Dependent Human Placental Cytotrophoblast Adhesion and Invasion In Vitro.

BACKGROUND: Anti-α4ß7 (Vedolizumab) treats inflammatory bowel disease (IBD) by blocking the interaction between integrin α4ß7 on leukocytes and mucosal addressin cell-adhesion molecule-1 (MAdCAM-1) on the gut endothelium. Women with IBD often require continuing biologic therapy during pregnancy to avoid disease flare. To date, there have been no reports of an increase in adverse events with Vedolizumab use during pregnancy. Notably, integrins play a major role in human placental development during pregnancy. It is unknown whether Vedolizumab disrupts placental cell (cytotrophoblast) invasion and/or adhesion by blocking interactions with MAdCAM-1. We therefore investigated human placental expression of MAdCAM-1, the role of MAdCAM-1/α4ß7 interactions in cytotrophoblast invasion/adhesion in vitro, and whether Vedolizumab administration in vivo alters the placental structure. METHODS: Histological sections of placentas from normal pregnancies were evaluated for MAdCAM-1 expression by immunofluorescence. The impacts of Vedolizumab or anti-integrin ß7 on human cytotrophoblast invasion and adhesion were assessed. Histology results from term placentas of 2 patients with IBD receiving Vedolizumab were compared to those of untreated healthy controls. RESULTS: Placental MAdCAM-1 expression was predominantly associated with invading extravillous cytotrophoblasts at the maternal-fetal interface. Treatment of isolated primary cytotrophoblasts with Vedolizumab or anti-integrin ß7 significantly reduced Matrigel invasion, adherence to a MAdCAM-1-coated substrate, and interactions with HuT-78 cells. Placentas from 2 Vedolizumab-treated patients with IBD exhibited pronounced pathologic features as compared to healthy control specimens. CONCLUSIONS: This study revealed a previously unrecognized role for α4ß7 and MAdCAM-1 in human placentation. More clinical and histological data from Vedolizumab-treated pregnant patients will be necessary to determine whether this medication poses any risk to the mother and fetus.

Relief of tumor hypoxia unleashes the tumoricidal potential of neutrophils.

Polymorphonuclear neutrophils (PMNs) are increasingly recognized to influence solid tumor development, but why their effects are so context dependent and even frequently divergent remains poorly understood. Using an autochthonous mouse model of uterine cancer and the administration of respiratory hyperoxia as a means to improve tumor oxygenation, we provide in vivo evidence that hypoxia is a potent determinant of tumor-associated PMN phenotypes and direct PMN-tumor cell interactions. Upon relief of tumor hypoxia, PMNs were recruited less intensely to the tumor-bearing uterus, but the recruited cells much more effectively killed tumor cells, an activity our data moreover suggested was mediated via their production of NADPH oxidase-derived reactive oxygen species and MMP-9. Simultaneously, their ability to promote tumor cell proliferation, which appeared to be mediated via their production of neutrophil elastase, was rendered less effective. Relieving tumor hypoxia thus greatly improved net PMN-dependent tumor control, leading to a massive reduction in tumor burden. Remarkably, this outcome was T cell independent. Together, these findings identify key hypoxia-regulated molecular mechanisms through which PMNs directly induce tumor cell death and proliferation in vivo and suggest that the contrasting properties of PMNs in different tumor settings may in part reflect the effects of hypoxia on direct PMN-tumor cell interactions.

Neutrophils Oppose Uterine Epithelial Carcinogenesis via Debridement of Hypoxic Tumor Cells.

Polymorphonuclear neutrophils (PMNs) are largely considered to foster cancer development despite wielding an arsenal of cytotoxic agents. Using a mouse model of PTEN-deficient uterine cancer, we describe a surprising inhibitory role for PMNs in epithelial carcinogenesis. By inducing tumor cell detachment from the basement membrane, PMNs impeded early-stage tumor growth and retarded malignant progression. Unexpectedly, PMN recruitment and tumor growth control occurred independently of lymphocytes and cellular senescence and instead ensued as part of the tumor's intrinsic inflammatory response to hypoxia. In humans, a PMN gene signature correlated with improved survival in several cancer subtypes, including PTEN-deficient uterine cancer. These findings provide insight into tumor-associated PMNs and reveal a context-specific capacity for PMNs to directly combat tumorigenesis.

Cyclosporin A impairs the secretion and activity of ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeat).

The protease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeat) cleaves multimers of von Willebrand factor, thus regulating platelet aggregation. ADAMTS13 deficiency leads to the fatal disorder thrombotic thrombocytopenic purpura (TTP). It has been observed that cyclosporin A (CsA) treatment, particularly in transplant patients, may sometimes be linked to the development of TTP. Until now, the reason for such a link was unclear. Here we provide evidence demonstrating that cyclophilin B (CypB) activity plays an important role in the secretion of active ADAMTS13. We found that CsA, an inhibitor of CypB, reduces the secretion of ADAMTS13 and leads to conformational changes in the protein resulting in diminished ADAMTS13 proteolytic activity. A direct, functional interaction between CypB (which possesses peptidyl-prolyl cis-trans isomerase (PPIase) and chaperone functions) and ADAMTS13 is demonstrated using immunoprecipitation and siRNA knockdown of CypB. Finally, CypB knock-out mice were found to have reduced ADAMTS13 levels. Taken together, our findings indicate that cyclophilin-mediated activity is an important factor affecting secretion and activity of ADAMTS13. The large number of proline residues in ADAMTS13 is consistent with the important role of cis-trans isomerization in the proper folding of this protein. These results altogether provide a novel mechanistic explanation for CsA-induced TTP in transplant patients.

Characterization of coding synonymous and non-synonymous variants in ADAMTS13 using ex vivo and in silico approaches.

Synonymous variations, which are defined as codon substitutions that do not change the encoded amino acid, were previously thought to have no effect on the properties of the synthesized protein(s). However, mounting evidence shows that these "silent" variations can have a significant impact on protein expression and function and should no longer be considered "silent". Here, the effects of six synonymous and six non-synonymous variations, previously found in the gene of ADAMTS13, the von Willebrand Factor (VWF) cleaving hemostatic protease, have been investigated using a variety of approaches. The ADAMTS13 mRNA and protein expression levels, as well as the conformation and activity of the variants have been compared to that of wild-type ADAMTS13. Interestingly, not only the non-synonymous variants but also the synonymous variants have been found to change the protein expression levels, conformation and function. Bioinformatic analysis of ADAMTS13 mRNA structure, amino acid conservation and codon usage allowed us to establish correlations between mRNA stability, RSCU, and intracellular protein expression. This study demonstrates that variants and more specifically, synonymous variants can have a substantial and definite effect on ADAMTS13 function and that bioinformatic analysis may allow development of predictive tools to identify variants that will have significant effects on the encoded protein.

A splice variant of ADAMTS13 is expressed in human hepatic stellate cells and cancerous tissues.

Although ADAMTS13, the von Willebrand factor (VWF)-cleaving protease, is expressed in a range of tissues, the physiological significance of tissue-specific ADAMTS13 alternative splicing isoforms have yet to be clarified. Screening a panel of human tissues and cell lines revealed a spliced ADAMTS13 transcript in hepatic stellate cells and a hepatoma cell line that retains the 25th intron. A nonsense codon within the intron truncates the protease, which gains 64 novel amino acids in lieu of both CUB domains. This isoform, while retaining VWF-cleaving capability, accumulates intracellularly and its biological inaccessibility may prevent its participation in regulating haemostasis and other physiologic functions.

Characterization of conformation-sensitive antibodies to ADAMTS13, the von Willebrand cleavage protease.

BACKGROUND: The zinc metalloprotease ADAMTS13 is a multidomain protein that cleaves von Willebrand Factor (VWF) and is implicated in Thrombotic Thrombocytopenic Purpura (TTP) pathogenesis. Understanding the mechanism of this protein is an important goal. Conformation sensitive antibodies have been used to monitor protein conformation and to decipher the molecular mechanism of proteins as well as to distinguish functional and non-functional mutants. METHODOLOGY/PRINCIPAL FINDINGS: We have characterized several antibodies against ADAMTS13, both monoclonal and polyclonal. We have used flow cytometry to estimate the binding of these antibodies to ADAMTS13 and demonstrate that antibodies raised against the TSP and disintegrin domains detect conformation changes in the ADAMTS13. Thus for example, increased binding of these antibodies was detected in the presence of the substrate (VWF), mainly at 37 degrees C and not at 4 degrees C. These antibodies could also detect differences between wild-type ADAMTS13 and the catalytically deficient mutant (P475S). The flow cytometry approach also allows us to estimate the reactivity of the antibody as well as its apparent affinity. CONCLUSIONS/SIGNIFICANCE: Our results suggest that these antibodies may serve as useful reagents to distinguish functional and non-functional ADAMTS13 and analyze conformational transitions to understand the catalytic mechanism.