Circulating Angiogenic Cell Dysfunction in Patients with Hereditary Hemorrhagic Telangiectasia

Liana Zucco,Ivana Kandic,Duncan J Stewart,Michael J Kutryk,Qiuwang Zhang,Marie E Faughnan,Michael A Kuliszewski,Francesco Bertolini

doi:10.1371/journal.pone.0089927

Liana Zucco, Ivana Kandic + Show 6 more

Open Access

https://doi.org/10.1371/journal.pone.0089927

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Abstract

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular disorder. Circulating angiogenic cells (CACs) play an important role in vascular repair and regeneration. This study was designed to examine the function of CACs derived from patients with HHT. Peripheral blood mononuclear cells (PBMNCs) isolated from patients with HHT and age- and gender-matched healthy volunteers were assessed for expression of CD34, CD133 and VEGF receptor 2 by flow cytometry. PBMNCs were cultured to procure early outgrowth CACs. Development of endothelial cell (EC) phenotype in CACs was analyzed by fluorescence microscopy. CAC apoptosis was assayed with Annexin V staining, and CAC migration assessed by a modified Boyden chamber assay. mRNA expression of endoglin (ENG), activin receptor-like kinase-1 (ACVLR1 or ALK1) and endothelial nitric oxide synthase (eNOS) in CACs was measured by real time RT-PCR. The percentage of CD34+ cells in PBMNCs from HHT patients was significantly higher than in PBMNCs of healthy controls. CACs derived from patients with HHT not only showed a significant reduction in EC-selective surface markers following 7-day culture, but also a significant increase in the rate of basal apoptosis and blunted migration in response to vascular endothelial growth factor and stromal cell-derived factor-1. CACs from HHT patients expressed significantly lower levels of ENG, ALK1 and eNOS mRNAs. In conclusion, CACs from patients with HHT exhibited various functional impairments, suggesting a reduced regenerative capacity of CACs to repair the vascular lesions seen in HHT patients.

Highlights

Hereditary hemorrhagic telangiectasia (HHT), an autosomal dominant vascular dysplasia, affects approximately 1 in 5–8000 people [1,2]
Most studies focussed on the exploration of endothelial progenitor cells (EPCs) for therapeutic angiogenesis or the examination of EPCs as potential biomarkers for cardiovascular disease, have used cells that are generated by the culture of peripheral blood mononuclear cells on fibronectin in vascular endothelial growth factor (VEGF)-containing medium [20,21,22]
After 2 washes with phosphate buffered saline (PBS), Peripheral blood mononuclear cells (PBMNCs) were directly analyzed by flow cytometry for the expression of CD34, CD133 and vascular endothelial growth factor receptor 2 (VEGFR2), or plated at a density of 0.756106/cm2 in human fibronectin-coated dishes or slides and cultured in endothelial cell basal medium supplemented with 20% human serum and various growth factors (EGM-2MV, Cambrex) to procure early outgrowth Circulating angiogenic cells (CACs)

Summary

Introduction

Hereditary hemorrhagic telangiectasia (HHT), an autosomal dominant vascular dysplasia, affects approximately 1 in 5–8000 people [1,2]. Most studies focussed on the exploration of EPCs for therapeutic angiogenesis or the examination of EPCs as potential biomarkers for cardiovascular disease, have used cells that are generated by the culture of peripheral blood mononuclear cells on fibronectin in vascular endothelial growth factor (VEGF)-containing medium [20,21,22]. These EPCs are not homogeneous but rather constitute a heterogenic population that mainly originates from myeloid hematopoietic cells [21,22,23,24,25,26]. We hypothesized that CAC function from patients with HHT is impaired, and can be demonstrated in a series of in vitro cell function assays

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Conclusion