HDL cholesterol is associated with PBMC expression of genes involved in HDL metabolism and atherogenesis

Background To reveal the association of plasma level of high density lipoprotein cholesterol (HDL-C) level with the transcript level of annotated genes in peripheral blood mononuclear cells (PBMC) and involved in HDL metabolism and atherogenesis at the absence of morphologically evident coronary stenosis. Methods Transcript levels of 63 genes in PBMC from 38 male patients 40-60 years without coronary atherosclerosis with widely varied HDL-C level were measured. The protein interactions were analyzed with STRING database. Results Among 22 HDL-related genes, the transcript levels for 10 genes (ABCA1, BMP1, CUBN, HDLBP, LCAT, LDLR, PRKACB, PRKACG, SCARB1 and ZDHHC8) negatively correlated with HDL-C, while positively for APOA1 gene. Among 41 atherosclerosis-prone genes, the transcript levels for 11 genes (CSF1R, CSF2RB, IL18R1, ITGAM, ITGB3, PRKCQ, SREBF1, TLR5, TLR8, TNFRSF1A and TNFRSF1B) negatively correlated with HDL-C only, not with LDL-C and plasma TG. The protein products efficiently interacted within each cluster while only two intersection nodes existed between clusters. Conclusions Coordinate regulation of cholesterol influx and efflux in PBMC in atherosclerosis-free subjects with widely varied HDL-C level is suggested. The decreased synthesis and transport of cholesteryl ester to the liver may contribute to hyperalphalipoproteinemia. HDL-C increase is associated with the decrease of expression of innate immunity and inflammation genes. Visualization of 22 responder genes is suggested to be useful in the validation of HDL functionality and atherogenesis even at the absence of morphologically evident coronary stenosis.


Introduction
Coronary heart disease is one of the major mortality causes around the globe (1). The major underlying effects are the disturbances of lipid metabolism and atherogenesis with the contribution of immunological and metabolic processes (2). For a long time, the primary goal in the treatment of atherogenic dyslipidemia was to decrease the low-density lipoprotein cholesterol (LDL-C) and to increase high-density lipoprotein cholesterol (HDL-C). The proatherogenic role of LDL was opposed to the atheroprotective role of HDL as cholesterol acceptors (3), thus forming the reverse cholesterol transport (RCT) from lipid-loaded macrophages to the liver (4). Also, HDL possesses many pleiotropic atheroprotective properties such as endothelium protection, antioxidant, anti-inflammatory, anti-apoptotic and anti-thrombotic effects (5,6). However, some genetic and clinical data challenged the atheroprotective significance of circulating HDL. The functional mutations of ABCA1 gene with low HDL-C levels did not increase the risk of coronary heart disease (7), while the increase of HDL-C level did not decrease the risk (8) and even increased it in some circumstances. GWAS studies revealed many single nucleotide polymorphisms associated with HDL content without any definitive conclusions on the association of HDL-C with coronary heart disease (9). The loss of the atheroprotective properties of HDL may be related to their functional properties, not with their concentration (10). The appearance of dysfunctional HDL may be caused by the loss of anti-inflammatory and antioxidant proteins with the concomitant appearance of pro-inflammatory proteins in HDL particles (11,12). Thus, the epidemiology, genetics, clinical and experimental data accumulated up to date do not unequivocally relate HDL-C as an anti-atherogenic factor.
To reveal the HDL role on the transcriptome level, we selected some genes with the expression in peripheral blood mononuclear cells (PBMC) (13). Among them, 22 genes involved in HDL metabolism (HDL cluster) were chosen. In addition, 41 genes involved in inflammation and other atherogenesisprone processes (atherogene cluster) were included based on the GWAS data on differentially expressed genes in PBMC in atherosclerosis versus control and RT-PCR data (13). In a pilot study with a small group of patients without coronary atherosclerosis, we com-pared the expression of HDL cluster genes in PBMC, HDL charge heterogeneity and cholesterol acceptor capacity of patient HDL in cholesterol efflux assay (14). The expression of several HDL-related genes, cholesterol efflux efficiency and concentration of preb-HDL significantly differed between hypo-and hyperalphalipoproteinemics (14). However, the joint consideration of the expression of atheroprotective and pro-atherogenic genes in relation to HDL-C level in the same group of patients is still lacking.
The aim of the present study is to reveal the association of plasma level of high-density lipoprotein cholesterol level with the transcript level of selected genes in peripheral blood mononuclear cells (PBMC) and involved in HDL metabolism and atherogenesis at the absence of morphologically evident coronary stenosis.

Patients and laboratory tests
Thirty-eight white Caucasian male patients at the age between 40 and 60 from the Moscow region were enrolled in the study ( Table I). The inclusion criteria were the absence of coronary deficiency and stenosis of coronary/carotid arteries verified by coronary angiography and ultrasonography, respectively, the absence of hypertension and diabetes, non-alcoholics, the absence of therapy by corticosteroids and contribute to hyperalphalipoproteinemia. HDL-C increase is associated with the decrease of expression of innate immunity and inflammation genes. Visualization of 22 responder genes is suggested to be useful in the validation of HDL functionality and atherogenesis even at the absence of morphologically evident coronary stenosis.
Klju~ne re~i: aterogeneza, ekspresija gena, HDL funkcionalnost, HDL i geni skloni aterogenezi, humani PBMC hypolipidemic agents for at least 3 months. The informed consent was obtained from each patient, and the local ethical committee approved protocol corresponding to the Helsinki declaration from 1975. Plasma lipid levels ( Table I) were measured by enzyme methods with Architect c8000 (Abbott, USA).

Analysis of interaction between protein products of genes belonging to two clusters
The analysis was performed with STRING database (15) with six kmeans clusters. The p-value for protein-protein interaction (PPI) enrichment is an indicator of interaction efficiency. The number of nodes and the number of edges are network characteristics.

Statistical analysis
Statistical analysis was performed using STATIS-TICA 8.0 data analysis software system (StatSoft, Inc. (2008)). We applied the Shapiro-Wilks W test to check the normality and nonnormally distributed data were log-transformed to approach a Gaussian distribution. The data are expressed as mean ± SD. The Primer GeneBank 5'-3' nucleotide sequence location efficiency Genes involved in atherogenesis   analysis of primer efficiencies was done with the REST software (16), and the relative expression of all target genes normalized by the geometric mean of three reference genes was used in the correlation analysis. We did linear regression and Pearson's correlation r tests between biochemical variables with transcript levels to determine the statistical significance and correlation. We used the Benjamini-Hochberg correction for multiple comparisons with the false discovery rate (FDR) value as 0.15 within the recommended range of 0.10 -0.20 (17). P-values < 0.05 were considered statistically significant.

Results
Expression of genes related to HDL metabolism PBMC expression level of 22 genes included in HDL cluster was measured by RT-PCR and significant associations of transcripts with plasma lipids are given in Table III. Significant associations existed between the transcripts of eleven genes and HDL-C. HDL-C level positively correlated with mRNA content of APOA1 gene, while negatively with transcripts of cholesterol transporters (ABCA1 and SCARB1), LDL receptor (LDLR), lecithin:cholesterol acyltransferase (LCAT), zinc finger DHHC-type containing 8 (ZDHHC8), bone morphogenetic protein 1 (BMP1), high density lipoprotein binding protein (HDLBP), cubilin (CUBN), protein kinase cAMP-activated catalytic subunit beta and gamma (PRKACB, PRKACG). Total cholesterol level also negatively correlated with transcript levels of the majority of the above-mentioned genes with the exception of LDLR and PRKACB genes. However, LDL-C level negatively correlated with transcripts of CUBN and HDLBP genes. Importantly, plasma TG level did not correlate with the transcript levels of HDL cluster genes.     Tyrosine-protein kinase that acts as cell-surface receptor for CSF1 and IL34 and plays an essential role in the regulation of survival, proliferation and differentiation of hematopoietic precursor cells, especially mononuclear phagocytes, such as macrophages and monocytes. Promotes the release of proinflammatory chemokines in response to IL34 and CSF1, and thereby plays an important role in innate immunity and in inflammatory processes.

IL18R1
-0.3898 (0.0170) Within the IL18 receptor complex, responsible for the binding of the proinflammatory cytokine IL18, but not IL1A nor IL1B (Probable). Contributes to IL18-induced cytokine production, either independently of SLC12A3, or as a complex with SLC12A3.

Integrin alpha-IIb/beta-3 recognizes the sequence H-H-L-G-G-G-A-K-Q-A-G-D-V in fibrinogen gamma chain.
ITGAM -0.4039 (0.0130) Integrin ITGAM/ITGB2 is implicated in various adhesive interactions of monocytes, macrophages and granulocytes as well as in mediating the uptake of complement-coated particles. It is identical with CR-3, the receptor for the iC3b fragment of the third complement component. It probably recognizes the R-G-D peptide in C3b. Integrin ITGAM/ITGB2 is also a receptor for fibrinogen, factor X and ICAM1.
Regulates transcription of the LDL receptor gene as well as the fatty acid and to a lesser degree the cholesterol synthesis pathway (By similarity). Binds to the sterol regulatory element 1 (SRE-1) (5'-ATCACCCCAC-3').

TLR5
-0.3649 (0.0240) Participates in the innate immune response to microbial agents. Mediates detection of bacterial flagellins. Acts via MYD88 and TRAF6, leading to NF-kappa-B activation, cytokine secretion and the inflammatory response.

TLR8
-0.5129 (0.0010) Key component of innate and adaptive immunity. TLRs control host immune response against pathogens through recognition of molecular patterns specific to microorganisms. Acts via MYD88 and TRAF6, leading to NF-kappa-B activation, cytokine secretion and the inflammatory TNFRSF1A -0.3718 (0.0220) Receptor for TNFSF2/TNF-alpha and homotrimeric TNFSF1/lymphotoxin-alpha. The adapter molecule FADD recruits caspase-8 to the activated receptor. The resulting death-inducing signalling complex (DISC) performs caspase-8 proteolytic activation which initiates the subsequent cascade of caspases (aspartate-specific cysteine proteases) mediating apoptosis.

Functional interactions between protein products of genes included in two clusters
The functional interactions between protein products of 22 genes, with PBMC expression level significantly associated with HDL-C level, were revealed by STRING database (15). The functional properties of proteins are included in Table III. Twentytwo nodes are involved in 50 interactions (Figure 1), which significantly exceeds eleven random interactions. Generally, nine from eleven genes from HDL cluster belong to Reactome Plasma lipoprotein assembly, remodelling, and clearance pathway (HSA-174824), based on minimal FDR value mentioned in STRING database. However, nine from eleven genes from the atherogene cluster belong to the Immune System pathway (HSA-168256). Two clusters intersect only at two nodes, namely CSFR1 (six edges) and CSF2RB (two edges) (Figure 1).

Discussion
The associations between plasma lipids and PBMC expression of 63 genes included in two clusters were studied for a cohort of 38 middle-aged male patients without coronary atherosclerosis with widely varied HDL-C. For the first time, the significant positive correlation between HDL-C and APOA1 expression and negative correlations of HDL-C and transcripts of ten HDL-related genes and eleven atherogenesis-prone genes even at the absence of morphologically evident coronary stenosis were revealed. The protein products efficiently interacted within both clusters, but only two-node intersections existed between them.
Monocytes are the main source of proinflammatory mediators at atherogenesis (34) that constitute one-fifth of the mononuclear cell fraction. PBMC expression of 41 genes included in the atherogene cluster was measured in the present study for 38 patients with a widely varied HDL-C level. HDL-C level negatively correlated with transcript content for eleven genes. The protein products of the some genes are of primary importance in inflammation and immune response, namely colony-stimulating factor receptors, TNF receptor superfamily members 1A and 1B, TLR5 and TLR8 receptors that induce the proinflammatory cytokine, and IL18 cytokine receptor that participates in innate immunity response and is a critical molecule in atherosclerosis-connected inflammation (35). Molecular mechanisms of the inhibition of expression by HDL remain unknown. HDL inhibits endothelial and monocyte expression of tissue factor (TF), the major coagulation factor, by inhibiting the activation of p38 MAPK (mitogen-activated protein kinase) and the repression of the PI3K (phosphoinositide 3-kinase) pathway responsible for TF expression (36). It should be noted that the negative correlation between HDL-C and ITGB3 and ITGAM gene transcripts observed here coincides with the inhibition of integrin and endothelial cell adhesion molecules by HDL (6,37). Overall, our data on the negative correlation between HDL-C and pro-inflammatory gene transcripts suggest the decay of atherogenesis-related inflammation with the increase of HDL-C. Besides, PBMC expression of atherogenesis-sensitive genes is suggested to be controlled only by HDL-C level, due to the lack of any association with LDL-C and plasma TG. Both conclusions are the major novelties of our study.
The limitation of our study is the expression profiling of only two gene clusters; the genome-wide transcriptome analysis may reveal some additional genes involved in atherogenesis-related pathways. Also, a detailed study of mRNA and protein expression and enzyme and receptor activities would confirm the metabolic traits suggested to be influenced by the genes in two clusters. However, both mRNA and protein expression of HDLBP/vigilin is upregulated similarly by intracellular cholesterol loading (27).
The positive correlation between HDL-C and APOA1 transcript existed among 22 HDL-related genes, while ten gene transcripts (ABCA1, BMP1, CUBN, HDLBP, LCAT, LDLR, PRKACB, PRKACG, SCARB1 and ZDHHC8) correlated negatively; they included six genes mentioned earlier (14). Four gene transcripts (CUBN, HDLBP, PRKACB, and PRKACG) negatively correlated with HDL-C in the present study. The significant decrease of HDLBP which expression is upregulated by intracellular cholesterol loading (27), thus confirms our earlier suggestion (14) on the decreased level of cholesterol in PBMC at hyperalphalipoproteinemia. Additionally, the decrease of the CUBN transcript, which codes the receptor for apoA-I and HDL (25), may result in an increase of HDL level. A decrease in the content of LCAT and SCARB1 transcripts may be suggested to result in the diminished flow of cholesteryl ester to the liver, thus contributing to hyperalphalipoproteinemia (14).
Functional relations between protein products of genes in two clusters include only two crossing genes. The existence of only two nodes may correspond to the limited number of synchronous changes in node activities in both clusters and the possibility of discordant changes of cluster functionality. Functional isolation of two clusters may underlie the possibility of non-coincident changes between the loss of HDL function and the gain of dysfunction (12). Multiple pleiotropic effects, namely anti-inflammatory activity, vasodilatory function, antiapoptotic activity, antioxidative activity and cholesterol efflux capacity (12), characterize HDL functions. It may be speculated further that therapy targeting these two nodes, for instance by drugs and\or microRNA, may influence both HDL functionality and atherogenesis. Interestingly, one of two nodes, namely CSF2RB mRNA, possesses the greatest pleiotropy across six cardiometabolic traits (38).

Conclusion
Coordinate regulation of cholesterol influx and efflux in PBMC in atherosclerosis-free subjects with widely varied HDL-C level is suggested. The decreased synthesis and transport of cholesteryl ester to the liver may contribute to hyperalphalipoproteinemia. HDL-C increase is associated with the decrease of expression of innate immunity and inflammation genes. Visualization of twenty-two responder genes is suggested to be useful in the validation of HDL functionality and atherogenesis even at the absence of morphologically evident coronary stenosis.