Endothelial Protective Properties of Low Molecular Weight Heparin (LMWH) Tinzaparin in Combination with Statin: A Novel Potential Therapeutic Tool in Preventing Cancer Metastasis?

Introduction

8.2 million people die from cancer worldwide every year. LMWH prolongs survival in newly-diagnosed, limited stage lung cancer. Interestingly, trials recruiting patients with other cancers have also demonstrated improved survival in patients with long life-expectancy but not in advanced malignancy, suggesting that the benefit may be mediated by metastasis prevention. LMWHs inhibit metastasis in vivo but may cause bleeding complications when used at effective doses. Consequently, LMWH is unlikely to be used for metastasis prevention unless bleeding side-effects can be urgently addressed. Enhanced endothelial barrier (EB) permeability is a hallmark of metastasis and permits the extravasation of circulating tumour cells into the tissues of the target organ.

Aims

To characterise LMWH Tinzaparin-mediated EB protection and underlying mechanisms and to optimise its cytoprotective properties in a manner that may reduce the associated bleeding risk.

Methods

EA.hy926 endothelial monolayers were exposed to LMWH (including a novel LMWH formulation comprised of a fraction of LMWH tinzaparin consisting of short polysaccharide chains, mean MW 2.8KDa), simvastatin (a lipid-lowering agent shown to exhibit EB protective properties) and either metastasis-associated agonists (thrombin & VEGF) or calcein blue-AM labelled metastatic tumour cells (DU145, prostate carcinoma). EB function was assessed through the measurement of the migration of an Evans blue-conjugated albumin solution/labelled tumour cells through the monolayers. The role of PAR-1 signalling in modulating permeability was assessed by flow cytometry and permeability assays using an anti-PAR-1 cleavage site antibody and PAR-1 activating/inhibitory peptides. Endothelial myosin light chain-2 diphosphorylation status (MLC-2; the key determinant of endothelial cell actin cytoskeleton contraction) was determined by SDS-PAGE and western blotting. The anticoagulant activity of the LMWHs was determined by calibrated automated thrombography (CAT) and plasma anti-factor Xa activity assays.

Results

Tinzaparin treatment prior to thrombin (IIa) and VEGF exposure significantly attenuated agonist-induced EB permeability in a concentration-dependent manner. This cytoprotective effect was observed within a clinically relevant concentration range (0-2IU/mL). Incubation of endothelial monolayers with LMWH also significantly reduced the migration of metastatic carcinoma cells (fluorescence of culture media containing labelled migrated tumour cells reduced to 73.9±5.7% of baseline; p<0.01).

IIa-induced permeability was found to be entirely mediated through PAR-1 cleavage. IIa-induced permeability was lost in the presence of an anti-PAR-1 receptor antibody and a PAR-1 signalling inhibitory peptide. Tinzaparin did not alter EA.hy926 PAR-1 expression and did not inhibit IIa-mediated PAR-1 cleavage but reduced IIa-induced MLC-2 phosphorylation and attenuated PAR-1 activating peptide-induced permeability, suggesting that the protective effect of LMWH is not mediated through an inhibition of IIa proteolytic activity (and is therefore independent of its anticoagulant activity).

The 2.8KDa LMWH fraction exhibited diminished anticoagulant activity relative to Tinzaparin but retained EB protective properties and attenuated tumour cell trans-migration (at a concentration equivalent to 0.5IU/mL, tumour cell migration was reduced to 76.4±4.7% of baseline and IIa-induced albumin permeability was attenuated to 66.9±8.9%).

Remarkably, the cytoprotective properties of Tinzaparin were significantly enhanced when combined with a statin. The combination of a sub-anticoagulant concentration of Tinzaparin (0.1IU/mL) and a clinically relevant concentration of simvastatin (20nM), attenuated EB permeability to a significantly greater extent that that induced by each agent alone (IIa-induced EB permeability was reduced to 7.9±0.2% of baseline (p<0.05)).

Conclusion

LMWH enhances the barrier function of endothelium in vitro. This effect is not linked to its anticoagulant activity. Strategies which permit using either non-anticoagulant LMWH or sub-anticoagulant LMWH concentrations in combination with other barrier-protective agents may represent a means of delivering this potentially anti-metastatic activity of LMWH to patients without conferring a bleeding risk.