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Gene Expression Patterns of Endothelial Cell Network Formation

ABSTRACT: Lamellar co-cultures of human aorta-derived smooth muscle cells (SMCs) cultured with cord-blood derived endothelial cells (ECs) are an in vitro model for tissue engineered blood vessels and in-growth of endothelial cells into cardiovascular repair devices. The ratio of endothelial cells to smooth muscle cells can control the EC pattern of growth and differentiation. Low density ECs form capillary-like networks in co-culture with SMCs whereas high density ECs become confluent across the top surface of the SMCs. To understand the molecular mechanisms that govern network formation, used microarray analysis of ECs purified from 1) networks, 2) confluent layers compared to 3) ECs in monoculture.

ORGANISM(S): Homo sapiens

PROVIDER: GSE30527 | GEO | 2017/03/01

SECONDARY ACCESSION(S): PRJNA143307

REPOSITORIES: GEO

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Project description:Coronary artery disease is the leading cause of mortality in women. Abnormal growth of smooth muscle cells and endothelial damage contributes to the thickening of the blood vessels and vascular occlusion. Estradiol prevents vascular remodleing by promoting EC growth and inhibiting SMC growth. Recent studies suggest that circulating (bone marrow derived) progenitor cells also contribute to the vascular remodeling process. Whether estradiol could, in part, induce its protective actions by inhibiting P-SMC growth remains unknown. In this study, progenitor smooth muscle cells (P-SMCs) and endothelial (P-ECs) grown from CD34+ progenitor cells (PC) magnetically separated from human cord blood derived mononuclear cells. P-SMC and P-EC cell colonies were grown and characterized for SMC and EC markers. Cultured cells were further used to assess the effects of 17β-estradiol (E2; 10nM) on their growth and gene expression. Briefly, P-SMCs or P-ECs were treated with or without 10 nM E2 for 0, 4 or 30 hours in DMEM-F12 supplemented with 2.5% FCS (steroid-free; SF). Subsequently, total RNA was extracted using RNeasy Mini Kit (QIAGEN) processed for gene analysis. Treatment of P-SMCs with E2 inhibited 2.5% SF-serum -induced growth (DNA synthesis and cell number), whereas, E2 induced cell growth in P-ECs. The growth effects of E2 in P-SMCs and P-ECs were mimicked by ER-α agonist and blocked by ER-α antagonist. The differential effects of E2 on P-SMC and P-EC growth were also reflected in ERK1/2 and Akt phosphorylation and in positive and negative regulators of cell cycle. Gene analysis revealed that P-SMCs expressed SMC alpha actin (ACTA2) where as P-ECs expressed CD34, FLT1 and vWF, confirming the differentiation of progenitor cells to SMCs and ECs. Treatment with E2 for 30 hours regulated 1791 in P-SMCs and 220 in P-ECs, with 36 that were common. Volcano analysis showed that treatment with E2 for 30 hours differentially expressed multiple genes, importantly, estradiol significantly upregulated antimitogenic pathways and downregulated proproliferative pathways in P-SMC and P-ECs, respectively. Contrasting effects of E2 were also observed in multiple ingenuity canonical pathways that were up or downregulated in P-SMCs and P-ECs. Findings from both growth and gene expression studies provide evidence that etradiol may induce its vascular protective actions by inhibiting P-SMC activity and improving P-EC activity. Interestingly, in perpheral blood estradiol decreasd the number of c-kit+ cells and increased CD34+ cells. Since these cells evolve into SMCs and ECs respectively, our findings provide evidence that estradiol may induce its anti-occlusive actions by differentially regulating P-SMC and P-EC activity and numbers.

Project description:Smooth muscle cells (SMCs) are important in a number of physiological systems and organs, including the cardiovascular system. The hallmark property of differentiated SMCs is the ability to contract, but contractile SMCs themselves show a range of phenotypes allowing prolonged tonic contraction in vascular smooth muscle or rapid phasic contraction in tissues such as bladder. Another distinctive characteristic, in contrast with terminally differentiated striated muscle cells, is that SMCs exhibit phenotypic plasticity. Vascular SMCs are able to modulate their phenotype along a continuum between a contractile phenotype, characteristic of healthy blood vessels, and a more proliferative âsyntheticâ phenotype, so-named for the enhanced synthesis and secretion of extracellular matrix components. Synthetic phenotype cells are found in a number of pathological situations such as atherosclerosis and arterial injury. We used mouse exon-junction (MJAY) arrays to gain insights into both the global contribution of alternative splicing events in re-shaping the transcriptome of dedifferentiating mouse aorta and bladder SMCs, and into the underlying regulatory mechanisms of the alternative splicing program. Affymetrix splice junction arrays (MJAY) were used to profile changes in both alternative splicing and transcript levels during the phenotypic modulation of smooth muscle cells when placed in culture. RNA extracted from intact aorta and bladder smooth muscle tissue was used for differentiated samples. For dedifferentiated, proliferative samples smooth muscle cells were enzymatically dispersed and grown in tissue culture for a week. Triplicate RNA samples were prepared from smooth muscle tissue of mouse aorta and bladder (differentiated) and from smooth muscle cells from each tissue cultured for 7 days (proliferative). The samples allowed comparison of alternative splicing (and other transcriptome) changes between differentiated and proliferative smooth muscle cell samples from two distinct types of smooth muscle cell, as well as allowing direct comparison of aorta (tonic smooth muscle) and bladder (phasic smooth muscle).

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Gene Expression Patterns of Endothelial Cell Network Formation

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