Co-expression network analysis

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highest ~0.12 quite low. The pathways with highest gene ratios comprise ‘Cytokine-cytokine receptor interaction’ and ‘MAPK signaling pathway’ (> 20 genes involved).

Also, the ‘NF-kappa B signaling pathway’, ‘Transcriptional mis-regulation in cancer’

and ‘Chemokine signaling pathway’ have comparably high gene counts (≤ 15 genes involved). Pathways with lower gene ratios (≤ 10 genes involved) include ‘Salmonella infection’, ‘PPAR-signaling pathway’, ‘Glutamatergic synapse’, ‘Ferroptosis’ and

‘Complement and coagulation cascade’.

Ferroptosis is a type of programmed cell death driven by loss of activity of the lipid repair enzyme glutathione peroxidase 4 (GPX4) and subsequent accumulation of lipid-based reactive oxygen species, particularly lipid hydroperoxides. This process is genetically and biochemically distinct from other forms of regulated cell death such as apoptosis, unregulated necrosis, and necroptosis.¹⁹⁵ Ferroptosis activation also often plays a regulatory role on growth of tumor cells in the human body which might explain the pathway involvement in ‘Transcriptional mis-regulation in cancer’. Ferroptosis could explain the high cell death rate in the 24h_PA cultures.

Figure 5.5.5.2 C.) shows pathways that are up-regulated in 24h_PA compared to 24h_OA stimulated monocytes. 226 DE-genes were taken under investigation.

Located pathways with highest gene counts and gene rations were defined as ‘MAPK signaling pathway’ and ‘Cytokine-cytokine receptor interaction’. DE-genes that either were up-regulated are integrated in ‘Fluid shear stress and atherosclerosis’ and

‘Colorectal cancer’, showing a gene ratio of 0.08. Further pathways up-regulated in 24h_PA stimulated cells encompass ‘NF-kappa B signaling pathway, ‘AGE-RAGE signaling pathway in diabetic complications’, ‘TGF-β signaling pathway’ and ‘Amino sugar and nucleotide metabolism’. The pathways are listed with decreasing gene counts and gene ratios. In this plot the p-values are all below 0.05 and thus pathways are most significant because the chance of randomly observing the same result is less than 5%.

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visualization and analysis of network graphs. A Pearson’s correlation coefficient cutoff of 0.88 was used to determine similarity between the individual expression profiles.

The set of the 50 most up-and 50 most down-regulated genes (highest/lowest fold change) between 24h_OA and 24h_Ctrl, 24h_PA and 24h_Ctrl and 24h_OA and 24h_PA cultured monocyte-derived cells were later filtered for p-values of ≤ 0.05 and a fold changes of ≥ 2 / ≤ -2 in one of the comparisons using the ANOVA table. Of the previously selected genes, 141 passed the filtering and where used as input for the network analysis.

The network was visualized in Cytoscape. The nodes within the network represent the expressed genes and their connection to eachother. The difference in expression relative to the overall mean, determined by the group fold change (GFC) of the respective condition, is indicated by color-coding of the respective nodes. Bluish colored nodes indicate that expression of the respective gene is down-regulated, while reddish nodes indicate that expression of the respective gene is up-regulated when compared to the overall expressed mean.

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Figure 5.5.6.1: Palmitic acid and oleic acid induced transcriptomic changes in myeloid-derived cells.

Visualization of co-expressed genes in monocytes after 24 hour treatment with oleic acid, palmitic acid and control medium. Differences in expression relative to the overall mean is indicated by color-coding of the respective nodes. The network is based on the top 50 highest and lowest differentially expressed genes (p-value ≤0.05) between OA and control, PA and control and OA and PA stimulated cells after 24 hours of culturing. The Pearson’s correlation coefficient cutoff was set to 0.88. The network consists of 136 nodes and 1339 edges.

Comparing the overall color distribution of the network, it is demonstrated that 24h_PA-primed cells (Figure 5.5.6.1 C.)) down-regulate all of the visualized genes that are up-regulated in the control cells or 24h_OA stimulated cells, and vice versa. This is only partially true when comparing the network coloration of 24h_OA-primed cells with the 24h_Ctrl (Figure 5.5.6.1 A.) & B.)). Here, at least some genes are regulated in the same direction and are just slightly up-regulated (rose color).

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Genes that are up-regulated in 24h_PA stimulated cells code for molecules involved in cell migration and proliferation (PDGFB, FOSB), for pro-inflammatory mediators and inflammatory response genes such as IL12B, CCL3L1, CCL4L2 and CXCL8, CCL3, HIVEP2 and genes involved in endocytosis and apoptosis like SH3D21, ADGRB1 and IL24. Additionally, the ABC1A gene, which codes for a protein functioning as cholesterol efflux pump in the cellular lipid removal pathway is up-regulated¹⁹⁶.

In case of 24h_Ctrl cultures, mainly genes that code for molecules involved in lipid and glycolipid binding, presentation and activation of T-cells (CD1A/B/E), involved in immunomodulatory and inflammatory processes (CCL17, CXCR1, CD4, IL1R2, HLA-DMB), in dendritic cell differentiation (CD207, CLEC4F, NDRG2), in the regulation of lipid metabolism (GPD1L) as well as proteins that play a role in adipocyte development and metabolism (STEAP4, HCAR2, SH3PXD2B), are strongly expressed.

In 24h_OA stimulated cells, genes which products are involved in cell adhesion and migration processes (FN1) and differentiation (PORCN), that participate in antigen presentation (CD1D, SCIMP), or are involved in leukocyte recruitment to inflammatory sides (CXCR2), such that participate in immune complex formation (C1QC, C1QA, CH25H), and such that function in immune response by triggering the production of constitutive inflammatory cytokines (TREM2) and other proteins that participate in the innate immune response (UBD), are up-regulated. As mentioned in the introduction, TREM2 is also associated with an atherosclerosis-specific population of inflammatory macrophages⁴⁶.

On the other hand, under the influence of 24h_OA also inflammation counter acting proteins, like CD300A and ASB13, are differentially expressed.

Further, genes are up-regulated, that code for molecules involved in metabolic processes, like FAR2, which catalyzes the reduction of saturated but not unsaturated C16 and C18 fatty acyl-CoA to fatty alcohols.

As previously mentioned, some few network genes that are up-regulated in both, 24h_Ctrl and 24h_OA stimulated cells could be identified. Proteins encoded by these genes comprise such involved in important metabolic pathways (MAOB-dopamine, SORD-glucose), or are involved in receptor-mediated endocytosis of lipoprotein and protein ligands (LRP6). MNDA is one of the proteins that play a role in the monocyte specific response to interferons. ZHX3, a protein associated with transcriptional repression is also differentially expressed. In both conditions genes coding for proteins

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involved in cell survival, like FAIM2, GIMAP6 and RIOX2 are elevated. Considering the coloration of the network nodes, it can be seen that most of the described genes are stronger expressed in the 24h_OA network (Figure 5.5.6.1 B.)) compared to the 24h_Ctrl network (Figure 5.5.6.1 A.)).

Gene and pathway information of this section were taken from http://www.pantherdb.org/ and https://www.genecards.org/ ^188,189.

5.6 Transcriptional analysis of monocyte-derived cells after wash-out of