Altered patterns of gene expression in endothelial cells in scleroderma
Authors: Howat S.L. , Abraham D. , Pearson J.D. 
Published: September 2009

Introduction  Scleroderma (systemic sclerosis, SSc) presents clinically as fibrosis of the skin but also involves fibrosis of blood vessels and internal organs, causing damage and complications that in the most severe forms lead to organ failure and death. The earliest detectable structural feature of SSc pathology appears to be endothelial cell damage. This leads to an inflammatory response with adhesion of leucocytes to the blood vessel walls, emigration and accumulation in the tissue. Paracrine factors secreted from activated endothelial cells have been implicated in the consequential fibroblast dysfunction and excessive deposition of extracellular matrix in lesional tissues (Denton et al. 1996). Activated lesional fibroblasts, which maintain their variant phenotype in culture for several passages, in turn, act upon the endothelial cells causing a ‘cross‐talk’ which perpetuates the SSc phenotype of both endothelial cells and fibroblasts. The aim of this work was to use an in vitro approach to identify the altered pattern of gene expression in endothelial cells induced by co‐culture with SSc lesional fibroblasts, which may reflect the phenotypic changes in endothelial cells in SSc.

Materials and methods  Human dermal microvascular endothelial cells (HMEC‐1, Ribeiro et al. 1995) were co‐cultured with dermal fibroblasts obtained from biopsies of lesional areas of the skin of patients with scleroderma or normal demal fibroblasts. Co‐cultures were carried out in six‐well transwell tissue culture plates. All cells were grown to confluence then fibroblasts with their conditioned medium were co‐cultured with HMEC‐1 for 2, 4, 6, 24 or 48 h. Total RNA was extracted from HMEC‐1, reverse transcribed, and expressed genes were identified using Atlas™ Nylon cDNA Expression Arrays (human broad range 1.2) (Clontech). Arrays were imaged using a Typhoon 9210 phosphorimager (Molecular Dynamics), and images were analysed using the AtlasImage™ 2.0 software. Selected mRNA levels were subsequently measured by real‐time PCR using a LightCycler™ (Roche).

Results  The human broad range 1.2 array has 1176 cDNAs plus nine housekeeping cDNAs and negative control cDNAs. The overall pattern of gene expression in HMEC‐1 co‐cultured with normal dermal fibroblasts (control) and HMEC‐1 co‐cultured with lesional SSc fibroblasts (diseased) was similar. In total, 32–49% of cDNAs were detectably expressed of which between 5 and 10% of cDNAs were apparently differentially expressed in diseased relative to control.

Ratios of diseased:  normal ranged from 11.7 to 0.09. Six genes of potential interest were selected from the 6‐h array and measured by real‐time PCR. Array results (at 6 h) showed that the leucocyte function‐associated molecule 1 alpha chain and caspase 10 were up‐regulated, and MMP‐11 was down‐regulated; connective tissue growth factor (CTGF), plasminogen activator inhibitor‐1 (PAI‐1) and endothelin‐2 (ET‐2) were expressed at similar levels in diseased and normal samples. When measured by real‐time PCR at all co‐culture time points, these genes were found to be either unchanged or down‐regulated in HMEC‐1 co‐cultured with SSc fibroblasts.

Discussion  We have identified the pattern of expression of 1176 cDNAs in HMEC‐1 co‐cultured with normal fibroblasts or with those from patients with SSc. These contain candidate genes which may be responsible for early vascular abnormalities in SSc. In future experiments, we will study the role of these candidate genes using in vitro models of the pathology of SSc.