A transcriptomic analysis of the EK1.Br strain of human fibroblastoid keratocytes: the effects of growth, quiescence and senescence.

There is a growing need within ocular research for well-defined cellular models of normal corneal biology. To meet this need we created and partially characterised a standard strain of human fibroblastoid keratocytes (EK1.Br) and demonstrated that phenotypic changes occur within these cells with replicative senescence in vitro. Using Affymetrix HG-U133A oligonucleotide arrays, this paper reports both a comprehensive analysis of the transcriptome of EK1.Br in the growing, quiescent and senescent states and a comparison of that transcriptome with those of primary corneal endothelium, lung fibroblasts and dermal fibroblasts grown under identical conditions. Data mining shows (i) that EK1.Br retain the characteristic transcriptional fingerprint of keratocytes in vitro (ii) that this phenotype can be distinguished from those of other 'fibroblasts' by groups of highly differentially expressed genes and (iii) that senescence induces a distinct dedifferentiation phenomenon in EK1.Br. These findings are contextualised into the broader literature on replicative senescence and are supported with a web-accessible and fully searchable public-access database (www.madras.cf.ac.uk/cornea).

Microarray analysis of senescent vascular smooth muscle cells: A link to atherosclerosis and vascular calcification.

Little is known about the senescent phenotype of human vascular smooth muscle cells (VSMCs) and the potential involvement of senescent VSMCs in age-related vascular disease, such as atherosclerosis. As such, VSMCs were grown and characterised in vitro to generate senescent VSMCs needed for microarray analysis (Affymetrix). Comparative analysis of the transcriptome profiles of early (14 CPD) and late (39-42 CPD) passage VSMCs found a total of 327 probesets called as differentially expressed: 149 are up-regulated in senescence and 178 repressed (p-value<0.5%, minimum effect size of at least 2-fold differential regulation, explore data at http://www.madras.cf.ac.uk/vsmc). Data mining shows a differential regulation of genes at senescence associated with the development of atherosclerosis and vascular calcification. These included genes with roles in inflammation (IL1beta, IL8, ICAM1, TNFAP3, ESM1 and CCL2), tissue remodelling (VEGF, VEGFbeta, ADM and MMP14) and vascular calcification (MGP, BMP2, SPP1, OPG and DCN). The microarray data for IL1beta, IL8 and MGP were validated by either, ELISA, Western blot analysis or RT-PCR. These data thus provide the first evidence for a role of VSMC senescence in the development of vascular calcification and provides further support for the involvement of senescent VSMCs in the progression of atherosclerosis.