Peptides do not prevent cleavage of endoglin to produce soluble endoglin

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Highlights

  • MMP14 cleaves endoglin at a specific peptide sequence to release soluble endoglin.

  • Short peptides that carry this peptide sequence were administered and soluble endoglin measured.

  • No effect on soluble endoglin was observed using JAR cells, primary explants, and primary HUVECs.

  • No effect on soluble endoglin was observed using HEK293Ts overexpressing MMP14 and endoglin.

  • Endoglin-like peptides do not inhibit soluble endoglin release.

Abstract

MMP14 cleaves membrane bound endoglin to produce soluble endoglin (sEng), an anti-angiogenic factor that causes endothelial dysfunction in preeclampsia. A recent publication proposed peptides with an amino acid sequence straddling the MMP14 cleavage site on endoglin decreases sEng release. This may be an exciting therapeutic approach and requires validation. We administered peptides to JAR cells, and primary placental explants and endothelial cells. The peptides had no effect on sEng production, and did not block sEng production in HEK293 with MMP14 and endoglin overexpressed. Peptides with an amino acid sequence encompassing the cleavage site do not prevent sEng production in vitro.

Introduction

Soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng) are anti-angiogenic factors that cause endothelial dysfunction seen in preeclampsia [1]. Adenoviral over-expression of sEng and sFlt1 in rats phenocopies the hallmarks of severe preeclampsia [1], [2]. Furthermore, serum sEng is markedly increased in women with preeclampsia [3]. Such studies highlight the critical role of sEng in severe preeclampsia.

Matrix metalloproteinase 14 (MMP-14; MT1-MMP) cleaves membrane bound endoglin at a specific peptide sequence close to the transmembrane domain, producing sEng [4]. We demonstrated that this mechanism plays a role in placental production of sEng [5]. Conceivably, blocking the cleavage of endoglin by MMP14 could represent a drug strategy to decrease sEng.

Recently, Valbuena-Diez et al. showed peptides with a sequence straddling the same amino acid sequence on transmembrane endoglin at the MMP14 cleavage site can reduce sEng release both in vitro and in vivo [6]. Conceivably, the peptides may work by blocking sEng release through competitive inhibition.

Given the therapeutic potential for such peptides to treat preeclampsia, we sought to independently validate this important finding in four separate in vitro systems including primary human tissues and a plasmid over-expression system.

Section snippets

Generation of endoglin peptides

Two sets of small peptides were ordered from Auspep (Pty Ltd, Victoria, Australia). One, therapeutic peptide, has an identical sequence to the MMP14 cleavage site on membrane bound endoglin (TSKGLVLP – cleavage site peptide). The second, a control peptide, is identical to a sequence in the extracellular portion of endoglin distant to the cleavage site. (GPRTVTVK – control peptide). The peptides were purified to >90% purity by Auspep, as previously published [6] (Fig. 1).

JAR cells

JAR cells

Results and discussion

We obtained two sets of peptides with the same amino acid sequence used in a report suggesting endoglin-like peptides could reduce sEng release [6]. The candidate therapeutic (named ‘cleavage site peptide’) is the same amino acid sequence that includes the specific glycine–leucine region on membrane endoglin where MMP-14 cleaves membrane bound endoglin to produce sEng (Fig. 1). The second peptide is identical in sequence as an extracellular region of endoglin distant from the MMP-14 cleavage

Ethics Statement

Human Ethics approval was obtained from the Mercy Health Human Research Ethics Committee.

Funding

The National Health and Medical Research Council of Australia provided salary (#1062418 to T.K, #628927 to N.H, #1050765 to S.T.) and project support (#1048707) and Australian Postgraduate Award to F.B.

Competing Interests

The authors have declared that no competing interests exist.

Acknowledgements

The authors acknowledge Clinical Research midwives Gabrielle Pell, Debra Jinks, Rachel Murdoch and Genevieve Christophers and the Obstetrics midwifery staff and patients at the Mercy Hospital for Women (Heidelberg) for their provision of placental tissue.

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