PROFILING AND CHARACTERIZING MICRORNAS IN PATIENTS WITH HEREDITARY HEMORRHAGIC TELANGIECTASIA

Abstract

Hereditary hemorrhagic telangiectasia (HHT) is a rare, autosomal dominant, vascular disorder that affects 1 in 5000 to 8000 individuals worldwide. HHT is characterized by vascular abnormalities, such as telangiectasias and arteriovenous malformations (AVMs). Telangiectasias are dilated skin and mucocutaneous blood vessels that are susceptible to spontaneous hemorrhage. AVMs are direct connections between arteries and veins, without intervening capillaries, and can cause significant right to left shunts. HHT arises from heterozygous mutations in at least 3 genes, ENG, ACVRL1, and SMAD4. Haploinsufficiency of their respective protein products perturb TGF signalling, affecting the angiogenic potential of endothelial cells (ECs) and the migratory capacity of monocytes, contributing to vascular malformations in HHT. MicroRNAs (miRs) are small non-coding RNAs that post-transcriptionally target mRNA transcripts. We aimed to profile and characterize miRs in HHT patient derived early endothelial progenitor cells (eEPCs) and peripheral blood mononuclear cells (PBMCs) to explore their potential role in HHT pathogenesis. EEPCs are early outgrowth ECs cultured from the mononuclear cell layer (buffy coat) of peripheral blood. They have been shown to augment angiogenesis in a paracrine manner by secreting various growth factors and are a widely accepted surrogate for studying EC function 28 HHT patients with confirmed mutations, and 25 controls were recruited. 40 ml of peripheral blood was collected for eEPC culture and PBMC isolation. Total RNA was isolated from PBMCs and eEPCs. PBMC and eEPC expression profiling was conducted with a human miR array analysis. Dysregulated miRs identified from the analyses were then validated with RT-qPCR. Significant differences were determined using a two-tailed t-test. Of the 800 miRs screened, 167 and 121 dysregulated miRs were identified in PBMCs and eEPCs, respectively. Selected PBMC miRs (MiRs-28-5p, -30b-5p, -361-3p and -374a-5p) and eEPC miRs (miRs-132-3p, -221-3p and -424-5p) were validated with RT-qPCR. PBMC miR-361-3p (p=0.025) known to target IGF1, and eEPC miR-132-3p (p=0.047) known to target SMAD2, were found to be significantly decreased compared to controls. Subsequently, IGF1 messenger RNA (mRNA) levels were significantly increased (p=0.005) in PBMCs of HHT patients compared to controls. Our results show dysregulation of select miRs in PBMCs and eEPCs from HHT patients. The finding of increased IGF1 mRNA and its possible relation to decreased miR-361-3p levels is both novel and exciting. This may represent a putative pathogenic mechanism involved in HHT, and may provide a unique miR therapeutic target for the treatment of clinical manifestations. National Institutes of Health (NIH)