A module of human peripheral blood mononuclear cell transcriptional network containing primitive and differentiation markers is related to specific cardiovascular health variables.

Leni Moldovan,Yang Xiang,Arunark Kolipaka,Mirela Anghelina,Nicanor I Moldovan,Kun Huang,Arshed Quyyumi,Taylor Kantor,William Malarkey,Peter J Mohler,Nima Ghasemzadeh,Desiree Jones,Enass Ramadan,Young-Sup Yoon

doi:10.1371/journal.pone.0095124

Abstract

Peripheral blood mononuclear cells (PBMCs), including rare circulating stem and progenitor cells (CSPCs), have important yet poorly understood roles in the maintenance and repair of blood vessels and perfused organs. Our hypothesis was that the identities and functions of CSPCs in cardiovascular health could be ascertained by analyzing the patterns of their co-expressed markers in unselected PBMC samples. Because gene microarrays had failed to detect many stem cell-associated genes, we performed quantitative real-time PCR to measure the expression of 45 primitive and tissue differentiation markers in PBMCs from healthy and hypertensive human subjects. We compared these expression levels to the subjects' demographic and cardiovascular risk factors, including vascular stiffness. The tested marker genes were expressed in all of samples and organized in hierarchical transcriptional network modules, constructed by a bottom-up approach. An index of gene expression in one of these modules (metagene), defined as the average standardized relative copy numbers of 15 pluripotency and cardiovascular differentiation markers, was negatively correlated (all p<0.03) with age (R2 = −0.23), vascular stiffness (R2 = −0.24), and central aortic pressure (R2 = −0.19) and positively correlated with body mass index (R2 = 0.72, in women). The co-expression of three neovascular markers was validated at the single-cell level using mRNA in situ hybridization and immunocytochemistry. The overall gene expression in this cardiovascular module was reduced by 72±22% in the patients compared with controls. However, the compactness of both modules was increased in the patients' samples, which was reflected in reduced dispersion of their nodes' degrees of connectivity, suggesting a more primitive character of the patients' CSPCs. In conclusion, our results show that the relationship between CSPCs and vascular function is encoded in modules of the PBMCs transcriptional network. Furthermore, the coordinated gene expression in these modules can be linked to cardiovascular risk factors and subclinical cardiovascular disease; thus, this measure may be useful for their diagnosis and prognosis.

Highlights

Circulating stem/progenitor cells (CSPCs) contribute to the maintenance of the normal functions of blood vessels and tissues and their repair and regeneration [1]
Module 1 To determine the physiological significance of the genes contributing to these co-expression patterns, we studied their individual relationships with the following characteristics of the blood donors: (i) age; (ii) the augmentation index (AIx) of their pulse, a surrogate measure of vascular stiffness [36]; (iii) blood pressure parameters; and (iv) body mass index (BMI)
We report several novel observations derived from an analysis of transcriptional activity in Peripheral blood mononuclear cells (PBMCs): (i) unlike GeneChips, which failed to reliably detect approximately half of the gene targets, qRT-PCR detected all 45 members of a panel of primitive and differentiation marker genes in all the tested human blood samples; (ii) based on their strong covariation, the target genes segregated into two major clusters, which exhibited the connectivity properties of modules in a bottom-up reconstituted hierarchical transcriptional network; (iii) one of modules contained most of the primitive and cardiovascular differentiation markers; (iv) this module correlated with several cardiovascular risk factors in the healthy blood donors, contributing to a cardiovascular-specific metagene; (v) the origin of genetic covariation was

Summary

Introduction

Circulating stem/progenitor cells (CSPCs) contribute to the maintenance of the normal functions of blood vessels and tissues and their repair and regeneration [1]. These cells may promote tumor growth by facilitating neovascularization or the development of tumor stroma [2]. A major obstacle to progress in this field has been a lack of consensus regarding the precise molecular markers that define these regenerative pathways [7] This problem is compounded by the limited accuracy and reproducibility of the current methods used to quantitate CSPCs, such as flow cytometry [8] and in vitro colony formation assays for ‘early’ [9] or ‘late’ [10] progenitor cells.

Methods

Results

Conclusion