Microarray Analysis of the Expression of Cultured Fibroblast Cells Following Stimulation by Nitric Oxide

Introduction: Pathologies of the tendon are relatively commonplace, yet current treatments for many of these problems are often suboptimal. Though most tendons have the ability to heal spontaneously after injury, the scar tissue that is formed is almost always mechanically inferior and therefore much less able to perform the functions of normal tendon, and is also more susceptible to further damage. Because the formulation of effective treatments for tendon injuries based on traditional tissue-level reparative procedures, surgical or otherwise, has presented such a problem to clinicians in the field, much research has been directed toward understanding the mechanisms of tendon healing at the molecular level. This has ultimately been in an effort to develop therapies to facilitate tendon healing through the use of individual molecules or groups of molecules known to have beneficial roles in the process. The healing cascade within the tendon is initiated, sustained, and eventually terminated by a large number and variety of molecules. Recent studies, however, have shown that the free radical nitric oxide (NO) has been shown to be a particularly important regulator of gene expression during many of these processes. Elsewhere NO has key roles in processes as varied as neuronal signalling and as an effector molecule for the immune system. It is produced by a class of enzymes known as the nitric oxide synthases (NOS), whose three isoforms all catalyse the conversion of L-arganine to NO and citrulline. NO has been found to be significantly upregulated in animal tendon damage models from the first day post-injury, and is thought to remain in abundance throughout the healing process. Though its important regulatory roles in tendon healing have been clearly shown, exactly which genes it exerts its effects on remains mostly unknown. The current study used microarray analyses to investigate the expression of 4,224 genes in cultured tendon fibroblast cells after stimulation with NO. Two different methods of NO delivery were used: addition of the chemical NO donor S-nitroso-N-acetyl-penicillamine (SNAP) to culture media, and transfection with the inducible nitric oxide synthase (iNOS) gene via an adenoviral vector. Following stimulation of fibroblast cells, RNA was extracted from each experimental and control group, and used for microarray expression analysis. Data analysis showed that several hundred genes had expression significantly upor downregulated compared to controls. Among these are several genes which may have important roles in tendon healing, including transcriptional and translational activators of growth factors, cellular migration signals, regulators of angiogenesis, morphogenic proteins, and extracellular matrix proteins.