We describe an mRNA profiling technique for determining differential gene expression that utilizes, but does not require, prior knowledge of gene sequences. This method permits high-throughput reproducible detection of most expressed sequences with a sensitivity of greater than 1 part in 100,000. Gene identification by database query of a restriction endonuclease fingerprint, confirmed by competitive PCR using gene-specific oligonucleotides, facilitates gene discovery by minimizing isolation procedures. This process, called GeneCalling, was validated by analysis of the gene expression profiles of normal and hypertrophic rat hearts following in vivo pressure overload.

We conducted large scale gene expression analysis of the response of macrophages to exposure to oxidized low density lipoprotein (Ox-LDL). Much of the vessel wall lesion of atherosclerosis is composed of macrophages that have become engorged with cholesterol. These resulting "foam cells" contribute to the progression of vascular disease through several pathways. As a potential model of foam cell formation, we treated THP-1 cells with 12-O-tetradecanoylphorbol 13-acetate to differentiate them into a macrophage-like phenotype and subsequently treated them with oxidized low density lipoprotein for various time periods. RNA from Ox-LDL treated and time-matched control untreated cells was hybridized to microarrays containing 9808 human genes. 268 genes were found to be at least 2-fold regulated at one or more time points. These regulation patterns were classified into seven clusters of expression profiles. The data is discussed in terms of the overall pattern of gene expression, the thematic classification of the responding genes, and the clustering of functional groups in distinct expression patterns. The magnitude and the temporal patterns of gene expression identified known and novel molecular components of the cellular response that are implicated in the growth, survival, migratory, inflammatory, and matrix remodeling activity of vessel wall macrophages. In particular, the role of nuclear receptors in mediating the gene expression modulation by Ox-LDL is highlighted.

Background-Normal myocardial development and the tissue response to cardiac stress are accompanied by marked changes in gene expression; however, the extent of these changes and their significance remain to be fully explored. We used cDNA microarrays for gene expression profiling in rat cardiac tissue samples to study developmental transitions and the response to myocardial infarction (MI). Methods and Results-Microarrays with rat cDNAs for 86 known genes and 989 anonymous cDNAs obtained by molecular subtraction (representational difference analysis) of mRNA from sham-operated and 6-week post-MI samples were used in 2-color hybridization experiments. Twelve known genes previously associated with myocardial development were identified together with 10 uncharacterized expressed sequence tags and 36 genes not previously associated with cardiac development. After MI, genes associated with myocardial stress and wound healing exhibited differences in magnitude and expression kinetics, and 14 genes not previously associated with MI were identified. In situ hybridization revealed mRNA localization characteristic of wound healing and vascular and cardiomyocyte reactivity. Conclusions-Tissue analysis of gene expression with cDNA microarrays provides a measure of transcriptional or posttranscriptional regulation and cellular recruitment. Our results demonstrate the complexity of gene regulation in the developing myocardium and show that cDNA microarrays can be used to monitor the evolution of the cardiac stress-inducible phenotype.

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Human mesenchymal stem cells (hMSC) are being administered by direct intramyocardial (IM) injection into patients with myocardial dysfunction with an objective to improve clinical status. However, surprisingly little attention has been directed to qualifying hMSC functionality beyond simple viability. In particular, the transit of hMSCs through a small-caliber needle lumen, the final fluidic pathway for all IM injection devices, may be especially prone to inducing unwarranted effects on cell function. This study evaluated the changes in clonogenicity, gene expression, and cytokine secretion that may be induced in hMSC (20 million/ml) by injection through a 26-gauge Nitinol needle at two different flow rates compared to noninjected control samples. Results indicated that hMSC viability and colony forming unit (CFU) formation was not altered by changes in injection rate, although a trend toward lower titers was noted at the higher flow rate, for the specific batch of hMSCs studied. The gene expression and cytokine analysis data suggest that delivering a suspension of MSCs through narrow lumen needles may marginally alter certain gene expression programs, but that such in vitro effects are transient and not translated into measurable differences in protein production. Gene expression levels of four cytokines (bFGF, SDF-1, SCF, VEGF) were significantly different at 400 µl/min, and that of all cytokines were significantly different at 1600 µl/min when compared to controls (p < 0.05). These changes were less pronounced (statistically insignificant for most cases, p > 0.05) and, in certain instances directionally opposite, at 72 h. However, no differences in the amounts of secreted bFGF, VEGF, or TGF-β were detectable at either of the two time points or flow rates. We infer that intramyocardial administration by transcatheter techniques is unlikely to interfere with the machinery required for cell replication or secretion of regulatory and other growth factors, which are the mainstays of MSC contribution to cardiac tissue repair and regeneration.Key words: Intramyocardial; Catheter; Delivery; Mesenchymal; Stem cells; Shear INTRODUCTIONlion (43), fulfilling the definition of a chronic epidemic. While advances in contemporary medical and surgical therapy improve the quality and duration of lives of many The leading cause of death in the US is heart disease (22), with ischemic processes constituting the predomipatients, each has its shortcomings, leading to a great deal of interest in cell-based therapies. nant pathogenic mechanism. A continuum of ischemic disease exists, initiated by acute infarction and perpetu-A variety of cell types have been utilized for cardiovascular repair (61), including autologous bone marrow ated both by additional ischemic insults and chronic changes in left ventricular geometry, bringing patients mononuclear cells (60) and their subpopulations (2,11, 52), autologous skeletal myoblasts (28), endothelial proto progressive left ventricular systolic dysfunction and the syndrome of congestive hear...