The Potential Role of MiRs-139-5p and -454-3p in Endoglin-Knockdown-Induced Angiogenic Dysfunction in HUVECs.

Hereditary hemorrhagic telangiectasia (HHT) is a rare genetic disease characterized by aberrant angiogenesis and vascular malformations. Mutations in the transforming growth factor beta co-receptor, endoglin (ENG), account for approximately half of known HHT cases and cause abnormal angiogenic activity in endothelial cells (ECs). To date, how ENG deficiency contributes to EC dysfunction remains to be fully understood. MicroRNAs (miRNAs) regulate virtually every cellular process. We hypothesized that ENG depletion results in miRNA dysregulation that plays an important role in mediating EC dysfunction. Our goal was to test the hypothesis by identifying dysregulated miRNAs in ENG-knockdown human umbilical vein endothelial cells (HUVECs) and characterizing their potential role in EC function. We identified 32 potentially downregulated miRNAs in ENG-knockdown HUVECs with a TaqMan miRNA microarray. MiRs-139-5p and -454-3p were found to be significantly downregulated after RT-qPCR validation. While the inhibition of miR-139-5p or miR-454-3p had no effect on HUVEC viability, proliferation or apoptosis, angiogenic capacity was significantly compromised as determined by a tube formation assay. Most notably, the overexpression of miRs-139-5p and -454-3p rescued impaired tube formation in HUVECs with ENG knockdown. To our knowledge, we are the first to demonstrate miRNA alterations after the knockdown of ENG in HUVECs. Our results indicate a potential role of miRs-139-5p and -454-3p in ENG-deficiency-induced angiogenic dysfunction in ECs. Further study to examine the involvement of miRs-139-5p and -454-3p in HHT pathogenesis is warranted.

Exploring Endothelial Colony-Forming Cells to Better Understand the Pathophysiology of Disease: An Updated Review.

Endothelial cell (EC) dysfunction has been implicated in a variety of pathological conditions. The collection of ECs from patients is typically conducted postmortem or through invasive procedures, such as surgery and interventional procedures, hampering efforts to clarify the role of ECs in disease onset and progression. In contrast, endothelial colony-forming cells (ECFCs), also termed late endothelial progenitor cells, late outgrowth endothelial cells, blood outgrowth endothelial cells, or endothelial outgrowth cells, are obtained in a minimally invasive manner, namely, by the culture of human peripheral blood mononuclear cells in endothelial growth medium. ECFCs resemble mature ECs phenotypically, genetically, and functionally, making them excellent surrogates for ECs. Numerous studies have been performed that examined ECFC function in conditions such as coronary artery disease, diabetes mellitus, hereditary hemorrhagic telangiectasia, congenital bicuspid aortic valve disease, pulmonary arterial hypertension, venous thromboembolic disease, and von Willebrand disease. Here, we provide an updated review of studies using ECFCs that were performed to better understand the pathophysiology of disease. We also discuss the potential of ECFCs as disease biomarkers and the standardized methods to culture, quantify, and evaluate ECFCs and suggest the future direction of research in this field.

MicroRNA-132-3p, Downregulated in Myeloid Angiogenic Cells from Hereditary Hemorrhagic Telangiectasia Patients, Is Enriched in the TGFß and PI3K/AKT Signalling Pathways.

BACKGROUND: Hereditary hemorrhagic telangiectasia (HHT) is a rare, autosomal dominant genetic disorder characterized by life-threatening vascular dysplasia. Myeloid angiogenic cells (MACs), alternatively called early endothelial progenitor cells or circulating angiogenic cells, do not directly incorporate into developing blood vessels, but augment angiogenesis in a paracrine manner. MAC dysfunction has been reported in HHT. MicroRNAs (miRNAs) regulate cellular function by modulating gene expression post-transcriptionally. To date, the role of miRNAs in HHT MAC dysfunction has not been documented. OBJECTIVE: The goal of this study was to comparatively profile miRNAs in HHT patient and control MACs to identify dysregulated miRNAs that may be responsible for the observed MAC dysfunction in HHT. METHODOLOGY/RESULTS: Twenty-three dysregulated miRNAs (twenty-one upregulated and two downregulated) in HHT MACs were identified with a TaqMan miRNA microarray. Pathway enrichment analysis showed that the dysregulated miRNAs were significantly enriched in pathways involved in HHT pathogenesis, such as the transforming growth factor ß (TGFß), phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT), and Hippo signalling pathways. Furthermore, miR-132-3p was determined to be significantly reduced in HHT MACs compared with controls by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Bioinformatic analysis revealed that miR-132-3p is significantly enriched in the TGFß and PI3K/AKT signalling pathways, targeting SMAD4, an effector of the TGFß signalling pathway and RASA1, a negative regulator of the PI3K/AKT signalling pathway, respectively. CONCLUSION: MiRNA dysregulation, specifically reduced expression of miR-132-3p, in HHT MACs was identified. The dysregulated miRNAs are significantly enriched in the TGFß, PI3K/AKT, and Hippo signalling pathways. These data suggest that alteration in miRNA expression may impair these pathways and contribute to MAC dysfunction in HHT.

Endoglin deficiency impairs VEGFR2 but not FGFR1 or TIE2 activation and alters VEGF-mediated cellular responses in human primary endothelial cells.

Hereditary hemorrhagic telangiectasia (HHT) is a genetic disease characterized by vascular dysplasia. Mutations of the endoglin (ENG) gene that encodes a co-receptor of the transforming growth factor ß1 signaling pathway cause type I HHT. ENG is primarily expressed in endothelial cells (ECs), but its interaction with other key angiogenic pathways to control angiogenesis has not been well addressed. The aim of this study is to investigate ENG interplay with VEGFR2, FGFR1 and TIE2 in primary human ECs. ENG was knocked-down with siRNA in human umbilical vein ECs (HUVECs) and human lung microvascular ECs (HMVEC-L). Gene expression was measured by RT-qPCR and Western blotting. Cell signaling pathway activation was analyzed by detecting phosphor-ERK and phosphor-AKT levels. Cell migration and apoptosis were assessed using the Boyden chamber assay and the CCK-8 Kit, respectively. Loss of ENG in HUVECs led to significantly reduced expression of VEGFR2 but not TIE2 or FGFR1, which was also confirmed in HMVEC-L. HUVECs lacking ENG had significantly lower levels of active Rac1 and a substantial reduction of the transcription factor Sp1, an activator of VEGFR2 transcription, in nuclei. Furthermore, VEGF- but not bFGF- or angiopoietin-1-induced phosphor-ERK and phosphor-AKT were suppressed in ENG deficient HUVECs. Functional analysis revealed that ENG knockdown inhibited cell migratory but enhanced anti-apoptotic activity induced by VEGF. In contrast, bFGF, angiopoietin-1 and -2 induced HUVEC migration and anti-apoptotic activities were not affected by ENG knockdown. In conclusion, ENG deficiency alters the VEGF/VEGFR2 pathway, which may play a role in HHT pathogenesis.

Non-Coding RNAs and Hereditary Hemorrhagic Telangiectasia.

Non-coding RNAs (ncRNAs) are functional ribonucleic acid (RNA) species that include microRNAs (miRs), a class of short non-coding RNAs (∼21-25 nucleotides), and long non-coding RNAs (lncRNAs) consisting of more than 200 nucleotides. They regulate gene expression post-transcriptionally and are involved in a wide range of pathophysiological processes. Hereditary hemorrhagic telangiectasia (HHT) is a rare disorder inherited in an autosomal dominant fashion characterized by vascular dysplasia. Patients can develop life-threatening vascular malformations and experience severe hemorrhaging. Effective pharmacological therapies are limited. The study of ncRNAs in HHT is an emerging field with great promise. This review will explore the current literature on the involvement of ncRNAs in HHT as diagnostic and pathogenic factors.

MicroRNA profiling of human myeloid angiogenic cells derived from peripheral blood mononuclear cells.

Human myeloid angiogenic cells (MACs), also termed early endothelial progenitor cells, play an important role in neovascularization and vascular repair. MicroRNAs (miRNAs) are a class of naturally occurring, noncoding, short (∼22 nucleotides), single-stranded RNAs that regulate gene expression post-transcriptionally. MiRNAs have been shown to regulate MAC function. A miRNA signature of MACs was described approximately a decade ago, and many new miRNAs have been discovered in recent years. In this study, we aimed to provide an up-to-date miRNA signature for human MACs. MACs were obtained by culture of human peripheral blood mononuclear cells in endothelial medium for 7 days. Using qPCR array analysis we identified 72 highly expressed miRNAs (CT value < 30) in human MACs. RT-qPCR quantification of select miRNAs revealed a strong correlation between the CT values detected by the array analysis and RT-qPCR, suggesting the miRNA signature generated by the qPCR array assay is accurate and reliable. Experimentally validated target genes of the 10 most highly expressed miRNAs were retrieved. Only a few of the targets and their respective miRNAs have been studied for their role in MAC biology. Our study therefore provides a valuable repository of miRNAs for future exploration of miRNA function in MACs.

MicroRNA signature of human blood mononuclear cells.

MicroRNAs (miRNAs) regulate a wide range of cellular processes and functions. Blood mononuclear cells (BMNCs) participate in the immune response, inflammatory reaction and angiogenesis. In 2010, a total of 157 miRNAs were quantified by RT-qPCR and a miRNA signature was determined for human peripheral BMNCs. With the advent of technologies such as RNA sequencing, many new miRNAs have been identified. This study was designed to provide an up-to-date miRNA signature for human BMNCs. Peripheral BMNCs were isolated by Ficoll density gradient centrifugation. Using the qPCR array assay, we identified 108 highly expressed miRNAs (Ct value < 30) in human BMNCs. Further validation of the array results by quantifying select miRNAs with RT-qPCR revealed a strong correlation between Ct values derived from array analysis and RT-qPCR, suggesting the array results presented in this study are accurate and reliable. Of note, the function of the majority of the highly expressed miRNAs we have identified has not yet been studied. Our findings may help direct further studies of the regulatory roles of miRNAs in BMNC function.

Decreased levels of miR-28-5p and miR-361-3p and increased levels of insulin-like growth factor 1 mRNA in mononuclear cells from patients with hereditary hemorrhagic telangiectasia 1.

Hereditary hemorrhagic telangiectasia (HHT) is a rare vascular disorder inherited in an autosomal dominant manner. Patients with HHT can develop vascular dysplasias called telangiectasias and arteriovenous malformations (AVMs). Our objective was to profile and characterize micro-RNAs (miRNAs), short noncoding RNAs that regulate gene expression posttranscriptionally, in HHT patient-derived peripheral blood mononuclear cells (PBMCs). PBMCs, comprised mostly of lymphocytes and monocytes, have been reported to be dysfunctional in HHT. A total of 40 clinically confirmed HHT patients and 22 controls were enrolled in this study. PBMCs were isolated from 16 mL of peripheral blood and purified for total RNA. MiRNA expression profiling was conducted with a human miRNA array analysis. Select dysregulated miRNAs and miRNA targets were validated with reverse transcription-quantitative polymerase chain reaction. Of the 377 miRNAs screened, 41 dysregulated miRNAs were identified. Both miR-28-5p and miR-361-3p, known to target insulin-like growth factor 1 (IGF1), a potent angiogenic growth factor, were found to be significantly downregulated in HHT patients. Consequently, IGF1 mRNA levels were found to be significantly elevated. Our research successfully identified miRNA dysregulation and elevated IGF1 mRNA levels in PBMCs from HHT patients. This novel discovery represents a potential pathogenic mechanism that could be targeted to alleviate clinical manifestations of HHT.

Dysfunction and Therapeutic Potential of Endothelial Progenitor Cells in Diabetes Mellitus.

Diabetes mellitus (DM) is a chronic, multifactorial metabolic disease whereby insulin deficiency or resistance results in hyperglycemia. Endothelial cells (ECs) form the innermost layer of the blood vessel and produce and release a variety of vasoactive substances and growth factors to regulate vascular homeostasis and angiogenesis. Hyperglycemia and insulin resistance can cause endothelial dysfunction, leading to vascular complications such as coronary artery disease, peripheral arterial disease, diabetic nephropathy, neuropathy and retinopathy. The detrimental effect exerted on ECs by hyperglycemia and insulin resistance underlines the importance of reparatory mechanisms in DM. Endothelial progenitor cells (EPCs), derived from bone marrow, have been recognized as endogenous cells involved in endothelial repair and new blood vessel formation. Initially isolated from a subset of circulating CD34+ mononuclear cells, EPCs were found to possess the ability to differentiate into ECs when cultured in vitro and incorporate into newly formed vessels upon transplantation in animal models of ischemia. Due to the low frequency of CD34+ cells in circulation, the vast majority of studies investigating EPC actions have used cells that are generated through the culture of peripheral blood mononuclear cells (PBMNCs) for 4 - 7 days in endothelial selective medium. These cells, mainly of myeloid hematopoietic cell origin, were termed "Early EPCs," of which, few expressed stem/progenitor-cell markers. Therefore, early EPCs were also termed "myeloid angiogenic cells" (MACs). When PBMNCs are cultured for over 2 weeks, early EPCs gradually diminish while so-called late EPCs appear. Late EPCs share phenotypic features with mature ECs and are therefore also termed blood-derived ECs; they will not be addressed in this review. MAC dysfunction has been observed in a variety of disease conditions including DM. In this article we review the activities and therapeutic potential of MACs in DM. We will interchangeably use "EPCs" and "MACs" to refer to the cells procured by culture of PBMNCs in EC selective medium for approximately 7 days.