Differential Expression of Circulating Plasma miRNA-370 and miRNA-10a from Patients with Hereditary Hemorrhagic Telangiectasia

Abstract

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant, vascular disorder that presents with telangiectases and arteriovenous malformations. HHT is a genetically heterogeneous disorder, involving mutations in endoglin (ENG; HHT1) and activin receptor-like kinase 1 (ACVRL1/ALK1; HHT2) genes that account for over 85% of all HHT patients. The current diagnosis of HHT patients remains at the clinical level, but many suspected patients do not have a clear HHT diagnosis or do not show pathogenic mutations in HHT genes. This situation has prompted the search for biomarkers to help in the early diagnosis of the disease. We have analyzed the plasma levels in HHT patients of selected micro-RNAs (miRNAs), small single-stranded RNAs that regulate gene expression at the transcriptional level by interacting with specific RNA targets. A total of 16 HHT1 and 17 HHT2 plasma samples from clinically confirmed patients and 16 controls were analyzed in this study. Total RNA was purified from plasma, and three selected miRNAs (miRNA-10a, miRNA-214, and miRNA-370), related to the pathobiology of cardiovascular diseases and potentially targeting ENG or ALK1, were measured by quantitative polymerase chain reaction. Compared with controls, levels of miRNA-370, whose putative target is ENG, were significantly downregulated in HHT1, but not in HHT2, whereas the levels of miRNA-10a, whose putative target is ALK1, were significantly upregulated in HHT2, but not in HHT1. In addition, the levels of miRNA-214, potentially targeting ENG and ALK1, did not change in either HHT1 or HHT2 patients versus control samples. While further studies are warranted, these results suggest that dysregulated plasma levels of miRNA-370 or miRNA-10a could help to identify undiagnosed HHT1 or HHT2 patients, respectively.

Keywords: activin receptor-like kinase 1 (ALK1); angiogenesis; arteriovenous malformations (AVMs); biomarker; bone morphogenetic protein (BMP); endoglin; hereditary hemorrhagic telangiectasia (HHT); microRNA; plasma; telangiectases; transforming growth factor beta (TGF-β).

Figure 1

Quantitative Reverse Transcriptase–Polymerase Chain Reaction (qRT-PCR) of miRNA-370. Total plasma RNA was isolated from HHT1 and HHT2 patients and control subjects. Relative expression levels of miR-370 were measured by qRT-PCR using miR-16 (A) or cel-miR-39-3p (B) as normalizers. The number of samples analyzed is indicated in parentheses. Symbols outside the box plot represent extreme values (°) and outliers (*) with their corresponding sample numbers. Experiments were performed in triplicates. (* p < 0.05; ** p < 0.01; ns, not significant).

Figure 2

qRT-PCR of miRNA-10a and miRNA-214. Total plasma RNA was isolated from HHT1 and HHT2 patients and control subjects. Relative expression levels of miRNA-10a (A,B) and miRNA-214 (C,D), measured by qRT-PCR using miR-16 (A,C) or cel-miR-39-3p (B,D) as normalizers. Symbols outside the box plot represent extreme values (°) and outliers (*) with their corresponding sample numbers. The number of samples analyzed is indicated in parentheses. Experiments were performed in triplicates. (* p < 0.05; ns, not significant).

Figure 3

Hypothetical model for the involvement of miRNA-370 and miRNA-10a in HHT. Top, in healthy subjects, basal levels of miRNA-370 and miRNA-10a are predicted to target endoglin, ALK1, as well as the VEGF/VEGFR pathway, thus regulating angiogenesis. Bottom, in HHT patients, heterozygous mutations lead to a deficient expression of endoglin (HHT1) or ALK1 (HHT2), associated with increased levels of VEGF and the dysregulated expression of miRNA-370 and miRNA-10a, resulting in abnormal angiogenesis. Treatment with antibodies to VEGF (bevacizumab) contributes to angiogenesis normalization. ALK1: activin receptor-like kinase 1; VEGF: vascular endothelial growth factor; VEGFR: VEGF receptors.