June 25, 2014

Probing Extracellular Vesicles’ Role in Cancer and Other Diseases

Microparticles provide window into endothelial cell function, thrombosis, malignancy

APS-Image-690-x-380

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By Keith McCrae, MD

All cells constitutively release submicron vesicles, termed “extracellular vesicles” (EV). EV with diameters greater than approximately 400 nm are generally referred to as “microparticles,” while EV less than 100 nm in diameter are usually termed “exosomes.” Most microparticles are derived by budding from the plasma membrane, while exosomes are derived from multivesicular bodies within endosomes.

EV are derived from all cell types, including platelets, endothelial cells, leukocytes and, in patients with cancer, malignant cells.

Due to their relatively large size, microparticles may be detected by flow cytometry and stained with specific antibodies to determine their cell of origin. While estimates of total circulating microparticle numbers vary, most studies estimate concentrations of approximately 1-3 x 106/mL of plasma.

Exosomes’ small size renders them below the limit of detection by flow cytometry, though they may be detected using biophysical approaches. Estimates of total plasma EV concentrations derived using such measurements are in the range of 108-109/mL.

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Since EV release from cells is enhanced by cellular activation or damage, elevated levels of circulating EV provide a biomarker for such processes.

EV and Antiphospholipid Antibody Syndrome

Our laboratory has long-standing interest in endothelial cell function, including the roles of endothelium in thrombosis and cancer. A particular area of interest is antiphospholipid antibody syndrome (APS), a disorder characterized by thrombosis and recurrent pregnancy loss in patients with antiphospholipid antibodies (aPL).

Antiphospholipid antibodies are a broad family of antibodies that includes lupus anticoagulants, anticardiolipin and anti-β2-glycoprotein I (β2GPI) antibodies, all of which may be detected in the clinical laboratory. Despite the confusing nomenclature, most pathologic aPL are actually directed against β2GPI, a phospholipid-binding protein that is abundant in plasma.

When activated or otherwise damaged, endothelium becomes dysfunctional, losing its normal anticoagulant ability and expressing procoagulant properties. Our previous work has demonstrated that aPL activate endothelial cells through a multireceptor pathway leading to activation of the protein complex NF-κB. The transcriptional activity of NF-κB results in decreased expression of anticoagulant, anti-inflammatory genes, and increased expression of procoagulant and proinflammatory genes. Our studies suggest that vascular activation is an important component of APS, as we observed significant elevations of circulating endothelial cell, platelet and tissue factor-expressing microparticles in 47 patients with APS compared with 150 healthy controls.

We have also explored the mechanisms of microparticle release by endothelial cells in response to aPL. We hypothesized that microparticle budding from cells would depend on rearrangements in the actomyosin cytoskeleton, which controls endothelial shape and membrane properties. We observed that upon exposure to aPL and their autoantigen, β2GPI, assembly of the actomyosin cytoskeleton was dramatically stimulated. Cytoskeletal assembly was dependent on phosphorylation of the myosin regulatory light chain (RLC) and was required for enhanced release of microparticles. Inhibitors of RLC phosphorylation blocked microparticle release from cultured endothelial cells exposed to aPL.

Conclusions and Research Directions

Taken together, these findings allow us to draw several conclusions. First, the elevation of circulating microparticles in patients with aPL suggests ongoing vascular activation, even in the absence of active thrombosis. Second, the release of microparticles from endothelial cells is critically dependent on assembly of the actin cytoskeleton, and the microparticles’ release thereby may be blocked by Rho kinase inhibitors, which have been suggested as potential therapeutics for patients with cancer. Third, the increased expression of tissue factor by microparticles from APS patients suggests that they may contribute directly to thrombosis development.

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EV also contain proteins, messenger RNA (mRNA) and small RNA species, particularly microRNA (miRNA), that may be freely transferred from the cell of origin to other cells. The content of EV varies dynamically according to the cell of origin and the nature of the stimulus that enhances their release.

To better understand the role of EV in disease, we have expanded our studies to include a detailed analysis of the biochemical and molecular composition of EV isolated from patients, including their content of specific proteins, mRNA and miRNA. Though our initial studies focused on APS, we have broadened our focus to include EV from patients with glioblastoma multiforme as well as pancreatic and other gastrointestinal cancers.

Proteomic and genomic analyses of tumor-derived circulating EV from patients with cancer may provide an opportunity to noninvasively obtain a “molecular snapshot” of the tumor in real time, providing information useful in designing therapies, determining prognosis, and monitoring molecular responses to standard and experimental agents.

Dr. McCrae is Director of Taussig Cancer Institute’s Benign Hematology Program, and a staff member of the Department of Hematologic Oncology and Blood Disorders and the Department of Cellular and Molecular Medicine. He can be reached at mccraek@ccf.org or 216.445.7809.

Images by Venkaiah Betapudi, PhD

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