Concentrations of low-density lipoprotein (LDL) above 0.8 mg/ml have been associated with increased risk for cardiovascular diseases and impaired endothelial functionality. Here, we demonstrate that high concentrations of LDL (1 mg/ml) decreased NOS3 protein and RNA levels in primary human endothelial cells. In addition, RNA sequencing data, in particular splice site usage analysis, showed a shift in NOS3 exon-exon junction reads towards those specifically assigned to nonfunctional transcript isoforms further diminishing the functional NOS3 levels. The reduction in NOS3 was accompanied by decreased migratory capacity, which depends on intact mitochondria and ATP formation. In line with these findings, we also observed a reduced ATP content. While mitochondrial mass was unaffected by high LDL, we found an increase in mitochondrial DNA copy number and mitochondrial RNA transcripts but decreased expression of nuclear genes coding for respiratory chain proteins. Therefore, high LDL treatment most likely results in an imbalance between respiratory chain complex proteins encoded in the mitochondria and in the nucleus resulting in impaired respiratory chain function explaining the reduction in ATP content. In conclusion, high LDL treatment leads to a decrease in active NOS3 and dysregulation of mitochondrial transcription, which is entailed by reduced ATP content and migratory capacity and thus, impairment of endothelial cell functionality.
Diet plays a crucial role in the development and prevention of cardiovascular diseases. A diet high in saturated fat increases the risk of heart disease and stroke. It is estimated to cause about 31% of coronary heart disease and 11% of stroke worldwide. Cholesterol is carried through our blood by particles called lipoproteins: high-density lipoprotein (HDL) and low-density lipoprotein (LDL). HDL cholesterol reduces the risk of cardiovascular disease as it carries cholesterol away from the bloodstream. High levels of LDL cholesterol lead to atherosclerosis increasing the risk of heart attack and ischemic stroke. Already in 2002, Minamino et al. demonstrated that human atherosclerotic lesions contain vascular endothelial cells with senescence-associated phenotypes [
Human primary endothelial cells were cultured in endothelial basal growth media (EBM) from Lonza, supplemented with hydrocortisone (1
For isolation of total RNA, cells were lysed using TRIzol (Thermo Fisher Scientific, Schwerte, Germany) and RNA was isolated according to the manufacturer’s specifications. RNA was subjected to a second purification step using the RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA concentrations were measured using a NanoDrop 2000c (Thermo Fisher Scientific, Schwerte, Germany), and RNA integrity was determined using a Bioanalyzer (Agilent, Waldbronn, Germany).
RNA sequencing data was obtained from quadruplicate total RNA samples. After DNase treatment, a library for sequencing was constructed using the TruSeq® Stranded mRNA Sample Preparation kit (Illumina), according to the Ribo-Zero protocol to remove ribosomal RNA. Subsequently, the libraries were sequenced using HiSeq3000 (Illumina) generating an average of 392 million single-end reads per sample. Library constructions and sequencing were performed at the Genomics and Transcriptomics Laboratory at the Biological Medical Research Centre (BMFZ) of the Heinrich-Heine University Düsseldorf. The quality of the reads was assessed using the tool FASTQC by Andrews (
Scripts used for this work are publicly available at
Total RNA was treated with RNAse-free DNase and reversed transcribed using SuperScript IV (Thermo Fisher Scientific, Schwerte, Germany) with random hexamers (pdN6) and oligo dT20 as primers. Relative transcript levels were determined by semiquantitative real-time PCR using the nuclear-encoded transcript for the ribosomal protein L32 (RPL32) as reference. The PCR reactions were done in a Rotor-Gene Q instrument (Qiagen, Hilden, Germany) using the SYBR Green qPCR Mastermix (Bimake, Munich, Germany) with the primer pairs listed below. All primer pairs for the analysis of nuclear transcripts were intron-spanning. For quantitation of mitochondrial transcripts, control reactions were performed with mock cDNAs, which were generated in a cDNA synthesis reaction without SuperScript IV. Relative expression was calculated as
Total DNA was isolated using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). DNA concentrations were measured using a NanoDrop 2000c (Thermo Fisher Scientific, Schwerte, Germany). Relative mtDNA levels were determined by semiquantitative real-time PCR using the single copy nuclear nucleoredoxin (NXN) gene as reference. PCR reactions were done in a Rotor-Gene Q instrument (Qiagen, Hilden, Germany) using the SYBR Green qPCR Mastermix (Bimake, Munich, Germany) with the primer pairs listed below. Relative mtDNA content was calculated as
Cells were lysed with JNK buffer (10 mM Tris-HCl, pH 7.5,150 mM NaCl, 2.5 mM KCl, 0.5% (
Cell migration assay was performed as described previously [
Cells were lysed with JNK buffer, and ATP was measured in total lysates as described previously [
Cells were treated with nonyl acridine orange and measured by flow cytometry as previously described [
Data were analyzed using unpaired Student’s
High LDL importantly contributes to the development and progression of cardiovascular diseases. However, the underlying molecular mechanisms how high LDL influences endothelial cell functionality are as yet poorly understood. In particular, detailed transcriptome analyses including the mitochondrial transcripts have not been addressed before. Therefore, we performed RNA deep sequencing of human primary endothelial cells after 7 days of treatment with high LDL. Prior to RNA sequencing, however, we validated that also in the current experimental setting, treatment with high LDL led to a reduction in NOS3 protein levels as described previously by us [
High LDL decreases NOS3 protein levels. Human primary endothelial cells were cultured in the standard medium (con) or medium containing 1 mg/ml LDL (high LDL) for 7 days. Full-length NOS3 protein was detected by immunoblot; ERK1/2 served as a loading control. (a) Representative immunoblots. (b) Semiquantitative analysis. Data are
RNA sequencing data showed that the observed decrease in the NOS3 protein levels was accompanied by a decrease in the total amount of NOS3 mRNA reads. In particular, differential gene expression analysis indicated a significant decrease in the NOS3 RNA levels in high LDL-treated cells compared to the healthy control with an adjusted
NOS3 transcript isoforms. Depicted at the top is the NOS3 protein (NOS3 protein) with its functional domains (OXY: oxygenase domain; CaM: calmodulin-binding site; FMN: FMN recognition site; AE: autoinhibitory element; FAD: FAD recognition site; NADPH: NADPH recognition site). The corresponding full-length transcript (NOS3 fl mRNA) annotated in Ensembl (v.93) with numbered exons is shown in the green dotted box. The coding region (wide boxes) extends from exon 2 into exon 27. Nonfunctional transcripts, i.e., transcripts not coding for functional NOS3, are shown in the red box below. Skipping of exon 20 (NOS3
Relative NOS3 exon-exon junction expression. Comparison of relative expression of all exon-exon junctions from exon 1 to exon 18 (1 : 18), exon 18 to exon 27 (18 : 27), and exon-exon junctions indicative of alternative splicing from exon 14 onto exons 14A/B/C (14-14x) or skipping exon 21 (20-22). The exon-exon junction coverage was normalized per sample to the number of gapped reads within the sample and to gene expression; the
Exon-exon junction | Average normalized expression | ||
---|---|---|---|
con | High LDL | ||
1 : 18 | 3,740 | 3,153 | 0.00 |
18:27 | 3,949 | 4,876 | 0.02 |
14-14x | 0.009 | 0.010 | 0.83 |
20-22 | 0.002 | 0.024 | 0.16 |
In this analysis, all exon-exon junctions showing a significant decrease in the relative coverage upon LDL treatment were located upstream of exon 18. In contrast, a significant increase was found in the junctions downstream of exon 18 (Table
A reduction in the functional NOS3 levels due to an unhealthy treatment leads to a decrease in the NO levels. Since endothelial cell migration is NO-dependent [
High LDL decreases migratory capacity of endothelial cells. Human primary endothelial cells were cultured in standard medium (con) or medium containing 1 mg/ml LDL (high LDL) for 5 days. A wound was set, and wound width was determined directly afterwards (0 h) and two hours later (2 h). (a) Representative microscopic pictures. (b) Wound closure relative to the 0 h time point. Data are
To address the question whether the expression of genes known to be associated with cell migration capacity could substantiate our finding, we performed differential gene expression analysis. Indeed, the expression of the cell cycle controlling protein CDC42, which is involved in cell migration was reduced by 35% in cells under high LDL conditions (
High LDL treatment significantly reduces ATP content in endothelial cells. Human primary endothelial cells were cultured in standard medium (con) or medium containing 1 mg/ml LDL (high LDL) for 5 days, and ATP content was measured. Data are
The dependence of migratory capacity on the ATP content was further confirmed by treatment of endothelial cells with oligomycin—a specific inhibitor of the mitochondrial ATP synthase. Both migratory capacity and ATP content were decreased by approximately 60% (Suppl. Figure
As we found reduced migratory capacity as well as lower ATP content in primary human endothelial cells upon treatment with high LDL, we next investigated the effects of high LDL on mitochondrial DNA and RNA levels, as well as on mitochondrial mass. Therefore, we first performed an alignment of the sequencing data to the human reference genome. In control samples (con), around 95.5% of the total reads could be mapped to the nuclear and 4.5% to the mitochondrial genome (Table
Percentage of read numbers representing nuclear and mitochondrial transcripts. Shown are total read numbers for all individual biological replicates, i.e., RNAs isolated from human primary endothelial cells cultured in standard medium (con_1-4) or medium containing 1 mg/ml LDL (high_LDL-1-4) and the percentage of reads, which could be mapped to the nuclear or mitochondrial reference genome.
Sample | Nuclear (%) | Mitochondrial (%) | Total # of reads |
---|---|---|---|
con_1 | 96.0 | 4.0 | 325,725,044 |
con_2 | 95.8 | 4.2 | 319,428,178 |
con_3 | 93.9 | 6.1 | 335,842,395 |
con_4 | 96.3 | 3.7 | 325,882,327 |
high_LDL_1 | 88.9 | 11.0 | 328,183,964 |
high_LDL_2 | 88.1 | 11.9 | 327,355,990 |
high_LDL_3 | 89.0 | 11.0 | 331,832,849 |
high_LDL_4 | 89.0 | 11.0 | 331,650,314 |
The upregulation of mtRNAs upon high LDL treatment raised the question as to whether this is paralleled by an elevation in mitochondrial DNA (mtDNA) content. Corresponding to the increase in mtRNA content, high LDL-treated cells showed a significantly higher mtDNA content (Figure
High LDL increases mtDNA content. Human primary endothelial cells were cultured in standard medium (con) or medium containing 1 mg/ml LDL (high LDL) for 7 days. Total DNA was isolated, and mtDNA content was measured by semiquantitative real-time PCR using NXN as nuclear reference gene. Data are
The increase in mtRNAs and mtDNA could be indicative of a higher number in mitochondria. Therefore, we determined the expression of genes coding for the translocases of outer (TOMMs) and inner (TIMMs) mitochondrial membrane proteins as surrogate markers. Those transcripts, however, were either not regulated or expressed at lower levels upon high LDL treatment. Corresponding to the non- or downregulated mRNA transcript levels, analysis of the TIMM23 protein levels as a marker for mitochondria showed no significant difference between the two conditions (data not shown). Thus, an increase in overall mitochondrial number induced by high LDL treatment was rather unlikely. We substantiated this by measuring mitochondrial mass using nonyl acridine orange. As shown in Figure
Mitochondrial mass is not altered by high LDL treatment. Human primary endothelial cells were cultured in standard medium (con) or medium containing 1 mg/ml LDL (high LDL) for 7 days. Then, cells were incubated with nonyl acridine orange and analyzed by flow cytometry. Data are
As the mtDNA content was increased upon high LDL without a concomitant change in mitochondrial mass, we next investigated the expression of protein coding mtRNA transcripts and mitochondrial ribosomal RNAs. Therefore, the RNA sequencing data were again analyzed for this specific subset of transcripts. High LDL-treated cells displayed an increase in the expression of these mitochondrial transcripts (Table
Differential gene expression of mitochondrial transcripts after high LDL treatment. Comparison of the expression of mitochondrial protein coding genes and ribosomal RNAs between untreated cells and cells treated with high LDL. The L2FC (log 2-fold change) states the average difference in gene expression between both treatments. Positive L2FC values denote upregulation by high LDL; negative values denote downregulation. A Wald test from DESeq2 was used to calculate the significance of the change in the expression. The adjusted
Gene name | Ensembl gene ID | L2FC | Adjusted | |
---|---|---|---|---|
MT-RNR1 | ENSG00000211459 | 1.86 | ||
MT-RNR2 | ENSG00000210082 | 1.52 | ||
MT-ND6 | ENSG00000198695 | 1.67 | ||
MT-ND1 | ENSG00000198888 | 1.34 | ||
MT-ND4 | ENSG00000198886 | 1.29 | ||
MT-ND3 | ENSG00000198840 | 1.33 | ||
MT-CO1 | ENSG00000198804 | 1.34 | ||
MT-CO2 | ENSG00000198712 | 1.19 | ||
MT-ATP6 | ENSG00000198899 | 1.00 | ||
MT-CYB | ENSG00000198727 | 1.01 | ||
MT-ND2 | ENSG00000198763 | 0.90 | ||
MT-CO3 | ENSG00000198938 | 0.87 | ||
MT-ND4L | ENSG00000212907 | 1.04 | ||
MT-ND5 | ENSG00000198786 | 1.40 | ||
MT-ATP8 | ENSG00000228253 | 0.82 |
To validate our RNA sequencing data, the transcript levels of mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 2 (MT-ND2), cytochrome B (MT-CYB), cytochrome C oxidase III (MT-CO3), and the mitochondrial 12S RNA (MT-RNR1) were analyzed by real-time PCR. The first three are subunits of electron transport chain complexes I, III, and IV, respectively. All of the chosen transcripts were significantly increased after treatment with high LDL (Figure
Transcript levels of mitochondrial genes are increased after treatment with high LDL for 7 days. Human primary endothelial cells were cultured in standard medium (con) or medium containing 1 mg/ml LDL (high LDL) for 7 days. Semiquantitative real-time PCRs were performed for MT-ND2 (a), MT-CYB (b), MT-CO3 (c), and MT-RNR1 (d) using RPL32 as reference. Data are
Since the mtRNAs were upregulated, we next investigated whether the nuclear-encoded transcription factors, which are known to be mainly responsible for the transcription of mtDNA, are regulated by high LDL. Expression analysis of the transcripts coding for mitochondrial transcription factor A (TFAM), B1 (TFB1M), and B2 (TFB2M) by real-time PCR, however, did not indicate any significant differences (Figure
Transcript levels of nuclear-encoded transcription factors of mtDNA transcription are not regulated by high LDL treatment. Human primary endothelial cells were cultured in standard medium (con) or medium containing 1 mg/ml LDL (high LDL) for 7 days. Semiquantitative real-time PCRs were performed for TFAM (a), TFB1M (b), and TFB2M (c) using RPL32 as reference. Data are
After transcription, the mtRNA precursor transcripts are cleaved by RNase P and RNase Z at the 5
mtRNA precursor transcripts are not regulated by high LDL treatment. Human primary endothelial cells were cultured in standard medium (con) or medium containing 1 mg/ml LDL (high LDL) for 7 days. Semiquantitative real-time PCRs were performed for mtRNA precursor transcripts using RPL32 as reference. Data are
Although some proteins of the respiratory chain are encoded on the mitochondrial DNA, most of them are derived from nuclear genes. Undisturbed interplay of mitochondrial and nuclear-encoded proteins ensures efficient respiratory chain complex formation and consequently ATP synthesis [
In contrast to the overall increased mitochondrial transcript levels following high LDL treatment, differential gene expression analysis for 81 nuclear genes encoding proteins of the respiratory chain revealed that 32% of them were significantly downregulated and only 4% upregulated.
The increase in mitochondrial gene expression and decrease in one third of nuclear genes for respiratory chain proteins could, therefore, restrict efficiency of ATP production due to an imbalance in respiratory chain subunits preventing proper assembly. Thus, one could assume that the reduced ATP content seen in Figure
The major findings of our study are that treatment of human primary endothelial cells with 1 mg/ml LDL for seven days decreases the NOS3 protein levels, increases inactive NOS3 splice variants, and reduces mitochondrial functionality in endothelial cells, which results in dramatically reduced migratory capacity and thus, endothelial cell impairment.
A functional endothelial cell layer is important, since it not only regulates vascular tone but as a barrier also regulates the nutrition uptake of the surrounding tissue and protects against pathogens. Endothelial cells are in direct contact with the bloodstream and consequently the first cells affected by LDL. Here, we demonstrate that treatment of endothelial cells with high LDL leads to decreased levels of functional NOS3 protein and mRNA levels. Additionally, RNA sequencing analyses revealed an increase in inactive NOS3 splice variants. This is accompanied by an increase in the expression of all mitochondrially encoded transcripts. However, there was no increase in total mitochondria number, as shown at the RNA level as well as at the protein level. It was previously described that NOS3-deficient mice showed a dysfunctional mitochondrial
We previously demonstrated that those concentrations of LDL resulted in endothelial cell senescence and increased ROS formation. Thus, we hypothesize that cells try to compensate the increased cellular stress, caused by those unhealthy conditions, by upregulating the expression profile of mitochondrial genes, like MT-ND2 and MT-CO3. Mitochondrial encoded genes are all part of the respiratory chain complexes. Thus, the cells try to cope for energy to handle the unfavorable situation. However, the majority of proteins needed for functional complex formation within the respiratory chain are encoded in the nuclear genome. Our data demonstrate, however, that most of those nuclear-encoded genes are downregulated upon high LDL treatment. Thus, an imbalance in proteins needed for the respiratory chain complexes seems plausible. This in turn would disturb efficient complex formation, resulting in reduced ATP production, which subsequently impairs ATP-dependent processes like endothelial cell migration as we show here.
We demonstrate that high LDL concentrations lead to low NOS3 levels in primary human endothelial cells, which is paralleled by mitochondrial dysfunction. We found an increase in mtDNA copy number and mtRNA levels as a potential compensatory mechanism for an unfavorable situation. However, due to an expression imbalance between nuclear and mitochondrial encoded proteins of the respiratory chain, complex formation is most likely impaired resulting in a drastic reduction in ATP levels. Consequently, the migratory capacity of the endothelial cells is reduced, which would negatively affect several cardiovascular diseases.
The RNA sequencing data used to support the findings of this study have been deposited at ArrayExpress under accession number E-MTAB-7647. All other data are available upon request.
All authors have disclosed that they do not have any conflicts of interest.
Stefanie Gonnissen, Johannes Ptok, Judith Haendeler, Heiner Schaal, and Joachim Altschmied contributed equally to this work.
This work was in part supported by the following grants: Deutsche Forschungsgemeinschaft (HA2868/10-2 and HA2868/11-1 to J.H., SCHA909/8-1 to H.S., TI323/4-1 to J.T., and SFB1116-2 A04 to J.H. and J.A.) and Forschungskommission of the Medical Faculty, Heinrich-Heine-Universität Düsseldorf (2016-44) to J.H. and H.S.; S.G. is a former scholarship holder of the IRTG1902, P1; K.J. is a scholarship holder of the IRTG1902, P1; P.J. is a former scholarship holder of the IRTG1902, P2.
Supplementary Figure 1: oligomycin reduces migratory capacity and ATP content in primary human endothelial cells. Primary human endothelial cells were left untreated (con) or treated with 10