Early Transcriptomic Response to LDL and oxLDL in Human Vascular Smooth Muscle Cells.

Although nowadays it is well known that the human transcriptome can importantly vary according to external or environmental condition, the reflection of this concept when studying oxidative stress and its direct relationship with gene expression profiling during the process of atherogenesis has not been thoroughly achieved.The ability to analyze genome-wide gene expression through transcriptomics has shown that the genome responds dynamically to diverse stimuli. Here, we describe the transcriptome of human vascular smooth muscle cells (hVSMC) stimulated by native and oxidized low-density lipoprotein (nLDL and oxLDL respectively), with the aim of assessing the early molecular changes that induce a response in this cell type resulting in a transcriptomic transformation. This expression has been demonstrated in atherosclerotic plaques in vivo and in vitro, particularly in the light of the oxidative modification hypothesis of atherosclerosis.Total RNA was isolated with TRIzol reagent (Life Technologies) and quality estimated using an Agilent 2100 bioanalyzer. The transcriptome of hVSMC under different experimental conditions (1,5 and 24 hours for nLDL and oxLDL) was obtained using the GeneChip Human Gene 1.0 ST (Affymetrix) designed to measure gene expression of 28,869 well-annotated genes. A fixed fold-change cut-off corresponding to ± 2 was used to identify genes exhibiting the most significant variation and statistical significance (P< 0.05), and 8 genes validated by qPCR using Taqman probes.10 molecular processes were significantly affected in hVSMC: Apoptosis and cell cycle, extracellular matrix remodeling, DNA repair, cholesterol efflux, cGMP biosynthesis, endocytic mechanisms, calcium homeostasis, redox balance, membrane trafficking and finally, the immune response to inflammation. The evidence we present supporting the hypothesis for the involvement of oxidative modification of several processes and metabolic pathways in atherosclerosis is strengthen by the fact that gene expression patterns obtained when hVSMC are incubated for a long period of time in the presence of nLDL, correspond very much the same as when cells are incubated for a short period of time in the presence of chemically modified oxLDL. Our results indicate that under physiological conditions and directly related to specific environmental conditions, LDL particles most probably suffer chemical modifications that initially serve as an alert signal to overcome a harmful stimulus that with time might get transformed to a pathological pattern and therefore consolidate a pathological condition

Early Transcriptomic Response to LDL and oxLDL in Human Vascular Smooth Muscle Cells - Fig 3

<p>Validation of microarray results by quantitative RT-PCR (A) Gene expression of ABCA1, SPP1, ZNF292,IL33, CALCRL, PRC1, KIF4A and C3 determined by quantitative real-time RT-PCR. Bars represent means ± SD (n = 3). Relative gene expression levels, normalized to GAPDH expression. Values superscribed by asterisks (*) are significantly different from nLDL and oxLDL groups (B) Heatmap Expression profile (microarray data).</p

hVSMC showing differential time dependent (1 to 24h) gene expression when exposed to nLDL or oxLDL.

<p>hVSMC showing differential time dependent (1 to 24h) gene expression when exposed to nLDL or oxLDL.</p

Top molecular and cellular functions significantly affected by the internalization of nLDL or oxLDL carried out at different times correlated with the top canonical pathways involved.

<p>Top molecular and cellular functions significantly affected by the internalization of nLDL or oxLDL carried out at different times correlated with the top canonical pathways involved.</p

Hierarchical cluster analysis of the differentially expressed genes with more than 2-fold changed expression in one out of six groups (nLDL 1h, nLDL 5h, nLDL 24h, oxLDL 1h, oxLDL 5h and oxLDL 24h) compared to parental hVSMC cells.

<p>The dendrogram on the left indicates correlation between gene expression profiles. The columns in the middle show the normalized expression of each gene in pseudocolor scale (key in the upper).</p

Hierarchical cluster analysis of several receptors and their expression when the six experimental conditions used (nLDL 1h, nLDL 5h, nLDL 24h, oxLDL 1h, oxLDL 5h and oxLDL 24h) are compared to parental hVSMC cells.

<p>Hierarchical cluster analysis of several receptors and their expression when the six experimental conditions used (nLDL 1h, nLDL 5h, nLDL 24h, oxLDL 1h, oxLDL 5h and oxLDL 24h) are compared to parental hVSMC cells.</p

Internalization Assays and lipid peroxidation in VSMC cells.

<p>White boxes corresponding at internalization in VSMC cells of 7.5 μg/mL of nLDLs or oxLDLs DiI label for 0 (Control), 1, 5, and 24 h under medium minimal culture conditions. The magnitude of internalization shown on the left side in the y-axis as relative fluorescence units (RFU) at 530/590 nm (excitation/emission). The blue squares correspond to the level of peroxidation of media supernatant after the same conditions. The lipid peroxidation level is shown in the y-axis on the right side expressed as [μM] of malondialdehyde.</p

Summary of main changes observed in gene expression and functions of hVSMC as a result of exposition to oxLDLs.

<p>Genes found over- or under-expressed in comparison to a control condition are shown (red and green respectively), and the predicted activation or inhibition in orange and blue.</p