Jansen, Matthias (2012) Teil 1: "Studies on the extracellular matrix enzyme lysyl oxidase (LOX) in epithelial cells" Teil 2: "Gene expression signatures of circulating peripheral blood cells associated with early-onset coronary artery disease". PhD thesis, Universität zu Köln.
Teil 1: The aim of the first part of my PhD project was to investigate the role of the extracellular matrix enzyme lysyl oxidase (LOX) in epithelial cells and its potential implications for cancers of epithelial origin. Using two well-characterized epithelial cell lines, i.e. MDCK II and MCF-10A, an in vitro model system was established to study the function of LOX in epithelia. For the first time the presence of mature LOX protein was demonstrated within cytoplasmic fractions. In addition, the enzyme was found to be secreted from the cells and BAPN-inhibitable LOX enzymatic activity was detected in concentrated fractions of conditioned cell medium. This result suggests that epithelial cells can express catalytically active LOX. Notably, extracellular expression and enzymatic activity of LOX were significantly elevated in post-confluent compared to pre-confluent cell cultures. This finding may indicate that LOX expression is elevated in differentiated epithelia. Although large amounts of mature LOX protein were observed in cytoplasmic fractions by western blotting, it was not possible to recover intracellular BAPN-inhibitable LOX enzymatic activity. LOX protein expression was shown to increase during scattering of MDCK II cells after hepatocyte growth factor (HGF) treatment. This process recapitulates important cellular characteristics of tumor metastasis in vivo. The higher LOX protein level correlated with a two-fold increase of LOX mRNA in HGF-treated compared to control cells as revealed by quantitative PCR. Therefore, the scatter assay may represent a suitable model system to study the recently reported role of LOX during cancer progression in vitro. Two strategies were followed to generate stable MDCK II lines that express recombinant LOX under a CMV promoter in order to determine whether constitutive LOX over-expression can induce changes of the epithelial phenotype. The first strategy comprised transfection of LOX-EGFP constructs. However, this approach did not yield detectable amounts of recombinant protein. Steric hindrance of the relatively large EGFP-tag (30 kD) may be a reason for the failure to express LOX- EGFP fusion proteins. Nevertheless, one clone transfected with full-length LOX-EGFP displayed an elongated morphology that resembled mesenchymal cells. The second approach used LOX-V5 expression constructs. Neither intra- nor extracellular expression of LOX induced significant phenotypical/morphological changes in MDCK II cells. In addition, no increase of LOX enzyme activity was detected in either, cytoplasmic fractions or conditioned cell medium of stably transfected cell lines. The difficulties in expressing recombinant LOX in this study represents a general challenge of the field and may result from its biochemical properties as well as the lack of more specific and sensitive assays to reliably determine its catalytic activity. Even though preliminary, the results of this study can serve as a promising basis for future investigations with the goal to decipher the precise function of LOX in epithelial cells. Teil 2: The aim of the second part of my PhD project was to identify differential gene expression signatures in circulating blood cells that are associated with coronary artery disease. Using a microarray-based whole genome expression profiling approach the transcriptome of circulating peripheral blood cells from individuals diagnosed with atherosclerotic coronary artery disease (CAD) was analyzed. The study cohort was assembled from a large clinical database hosted by the Veterans Administration Pacific Islands Healthcare System (VAPHICS). Samples from patients with early myocardial infarction (MI) prior to age 50 and clinically diagnosed with CAD were compared to a healthy control group treated with the same cardiac-related medication. Gene expression profiles from whole blood mRNA samples that were depleted for β-globin transcripts was assessed by microarray analysis. A novel algorithm called LOTEST that was specifically designed to detect heterogenous and sparse signals embedded within gaussian noise, identified 1203 differentially expressed genes between patients and controls. Out of these, 195 genes were excluded from downstream analysis as they were represented by only two individuals within the patient group. The gene ontology databases „Protein Analysis Through Evolutionary Relationships“ (PANTHER) and „Ingenuity Pathway Analysis“ (IPA) were then applied to analyze functional grouping within the identified differential gene expression pattern. The analysis revealed over-representation of genes that are associated with inflammation and immune system function. A list of potential candidate genes with the most consistent expression pattern across the sample groups was confirmed by quantitative real-time PCR analysis and included up-regulated pro- inflammatory genes (PTX3, LGALS3, CAMP), down-regulated anti-inflammatory genes (IL12RB1, JAG1), decreased expression of genes that protect from auto-immunity (IL2RA, CCR7) and genes not previously implicated in immune system function (GSTT1, NGFR). The results of this study suggest persistent ongoing inflammation in CAD patients of the study cohort despite treatment with anti-inflammatory medication. The outcome of this small-scale study can serve as a basis for future investigations in order to discover new biomarkers and/or potential targets for drug development against so far unknown disease mechanism(s). Certainly, the realization of these tasks would require substantial epidemiological and basic research efforts.
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