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Genetic Ablation of Transmembrane Prolyl 4-Hydroxylase Reduces Atherosclerotic Plaques in Mice

Originally publishedhttps://doi.org/10.1161/ATVBAHA.121.316034Arteriosclerosis, Thrombosis, and Vascular Biology. 2021;41:2128–2140

Abstract

Objective:

Atherosclerosis is a key component of cardiovascular diseases. We set out to study here whether genetic ablation of P4H-TM (transmembrane prolyl 4-hydroxylase) could protect against atherosclerosis as does inhibition of the other 3 classical HIF-P4Hs (hypoxia-inducible factor prolyl 4-hydroxylases).

Approach and Results:

We generated a double knockout mouse line deficient in P4H-TM and LDL (low-density lipoprotein) receptor (P4h-tm−/−/Ldlr−/−) and subjected these mice to a high-fat diet for 13 weeks. The double knockout mice had less atherosclerotic plaques in their full-length aorta than their P4h-tm+/+/Ldlr−/− counterparts and also had lower serum triglyceride levels on standard laboratory diet and high-fat diet, higher levels of IgM autoantibodies against Ox-LDL (oxidized LDL), and significantly higher LPL (lipoprotein lipase) protein levels in white adipose tissue and sera. RNA-sequencing analysis revealed changes in expression of mRNAs in multiple pathways including lipid metabolism and immunologic response in the P4h-tm−/−/Ldlr−/− livers as compared with P4h-tm+/+/Ldlr−/−.

Conclusions:

Our data identify P4H-TM inhibition as a potential novel immuno-metabolic mechanism for intervening in the pathology of atherosclerosis, as hypertriglyceridemia is an individual risk factor for atherosclerosis, and IgM antibodies to Ox-LDL and increased lipoprotein lipase have been associated with protection against it.

Highlights

  • P4H-TM (transmembrane prolyl 4-hydroxylase) is a novel gene associated with atherosclerosis.

  • P4H-TM deficiency coincides with reduced serum triglyceride levels.

  • P4H-TM deficiency in combination with high-fat diet alters the levels of circulating atheroprotective autoantibodies.

  • P4H-TM deficiency co-occurs with increased levels of LPL (lipoprotein lipase) in white adipose tissue and sera.

  • P4H-TM inhibition offers a potential novel mechanism for intervening in the pathology of atherosclerosis.

Introduction

Atherosclerosis is a slow, progressive disease that affects arteries throughout the body. It is the key component in cardiovascular diseases, the number one cause of death globally, for which mechanisms remain to be discovered.1 The risk factors for atherosclerosis include elevated serum cholesterol levels, especially LDL (low-density lipoprotein), hypertension, diabetes, smoking, obesity, unhealthy diet, and family history.2 Current treatment targets these with statins, antihypertensive drugs, and diet and exercise interventions. Atherosclerosis is also an inflammatory disease in which immune mechanisms interact with metabolic risk factors to initiate, propagate, and activate the arterial lesions.1,3,4 Higher oxidative stress associates with atherosclerosis. When LDL retained in the subendothelial space becomes oxidized (Ox-LDL [oxidized LDL]), it acquires immunogenic properties. As a result different lipid peroxidation-derived structures, that are recognized as antigens by the immune system, are formed. These oxidation-specific epitopes, such as malondialdehyde, resemble those in bacteria and can be recognized by natural antibodies which are part of the innate immune system.5 The main natural immunoglobulins, IgM and IgG, are produced by the B cells and neutralize both pathogens and variety of self-antigens, including oxidation-specific epitopes. According to current understanding, IgMs mediate atheroprotection while the role of IgGs is controversial.5

HIF-P4Hs (HIF [hypoxia-inducible factor] prolyl 4-hydroxylases; also known as PHDs or EglNs) are enzymes that act as cellular oxygen sensors.6,7 When oxygen is available, they hydroxylate prolyl residues in the HIFα subunit, which earmarks it for proteasomal degradation.6 Decline in oxygen levels inhibits HIF-P4H activity and a transcriptionally active HIFαβ dimer is formed to upregulate the expression of several hundred genes to balance the oxygen supply and demand.6,8,9 One of the key processes regulated by HIF is the reprogramming of energy metabolism, namely downregulation of the O2-demanding mitochondrial oxidative phosphorylation and upregulation of nonoxygen-demanding glycolysis.8 This nevertheless comes at the expense of reduced ATP generation.8 Other key processes regulated by HIF are erythropoiesis and angiogenesis. In addition, HIF target genes regulate inflammatory and immunologic processes, cell proliferation and apoptosis, and balance matrix and barrier functions.6,9,10 HIF-P4Hs can be targeted with chemical inhibitors that stabilize HIF under normoxia. The first-in-class small-molecule HIF-P4H inhibitor has been approved for the treatment of renal anemia and several others are undergoing clinical trials.8,10,11 Of the 3 classical HIF-P4Hs, isoenzyme 2 (HIF-P4H-2/PHD2/EglN1) is the most abundant and the major one regulating HIFα stability.12,13 Large-spectrum genetic knockdown of HIF-P4H-2 in hypomorph mice (Hif-p4h-2gt/gt) results in reduced body weight and adiposity, lower serum cholesterol levels, reduced white adipose tissue (WAT) inflammation, improved glucose tolerance and higher levels of circulating Ox-LDL targeting autoantibodies,14,15 as well as protection against metabolic disorders such as fatty liver disease.16,17 Interestingly, tissue-specific inactivation of HIF-P4H-2 in adipose tissue only is sufficient to provide some of the beneficial metabolic outcomes.18,19 Moreover, crossing of the Hif-p4h-2gt/gt mice with LDL receptor mutant mice and exposing them to a high-fat diet (HFD) provided protection against atherosclerosis.15 HIF-P4H-1 deficiency reduced skeletal muscle O2 consumption, LDL-cholesterol levels, circulating immune cells, and glucose intolerance,20,21 although Hif-p4h-1−/− mice were associated with hepatic steatosis and liver-specific insulin resistance.22 Nevertheless, Hif-p4h-1−/−/Ldlr−/− mice on HFD were protected from atherosclerosis.21 Overexpression of HIF-P4H-3 in ApoE−/− mice showed increased atherosclerosis and enhancement of macrophages and smooth muscle cells associated with upregulation of the expression of ICAM-1 (intercellular adhesion molecule 1), VCAM-1 (vascular cell adhesion molecule 1), MCP-1 (monocyte chemoattractant protein 1), IL-1β (interleukin 1 beta), and TNF-α (tumor necrosis factor alpha), thus suggesting that HIF-P4H-3 inhibition was also associated with atheroprotection.23 Administration of a pan HIF-P4H inhibitor FG-4497 protected Ldlr−/− mice on HFD against atherosclerosis15 and was associated with reduced body weight, lower serum cholesterol levels, less WAT inflammation, and higher atherosclerosis-protective circulating autoantibody levels compared with vehicle treatment.15

P4H-TM (transmembrane prolyl 4-hydroxylase) is a fourth HIF-P4H possessing an unique EF-domain and a catalytic activity potentially regulated by Ca2+.24 It hydroxylates HIFα in vitro and its inhibition upregulated HIFα levels in cells, although it also hydroxylated HIFα after mutation of the prolines targeted by HIF-P4Hs 1-3, suggesting that it may have additional substrates.25 This is also suggested by the location of its catalytic domain within the endoplasmic reticulum, while HIFα is cytosolic or nuclear.25 The expression level of P4h-tm mRNA is highest in the brain and eye, followed by the kidney, adrenal medulla, and skeletal muscle.26,27 In humans, P4H-TM mutations associate with HIDEA syndrome, a neurological phenotype featuring hypotonia, hypoventilation, intellectual disability, dysautonomia, epilepsy, and eye abnormalities.28,29P4h-tm−/− mice present with upregulated erythropoiesis,30 early-onset age-related retinal and renal dysfunction,26 increased social behavior with reduced fear and anxiety27 and disturbed calcium signaling in the brain.31 In zebrafish, P4H-TM deficiency resulted in compromised kidney function and defects in basement membranes.32 Since very little is known about the possible contribution of P4H-TM to metabolic regulation, we set out to study whether P4H-TM inhibition, like that of other HIF-P4Hs, plays a role in atherosclerosis in mice. For that, we crossed P4h-tm−/− and Ldlr−/− mice and subjected the double knockouts to a HFD. At euthanization, the P4h-tm−/−/Ldlr−/− mice had less plaques in their full-length aorta, lower serum triglycerides, higher amount of circulating atheroprotective autoantibodies, and increased LPL (lipoprotein lipase) protein levels compared with the P4h-tm+/+/Ldlr−/− mice.

Materials and Methods

Data Access

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Animal Experiments

All the experiments were performed according to protocols approved by the National Animal Experiment Board of Finland, license numbers ESAVI-6154 and ESAVI-8179. The P4h-tm−/− mice were generated as described.26,30Ldlr−/− mice were purchased from the Jackson Laboratory and backcrossed with C57BL/6 N mice for 10 generations. The P4h-tm+/− mice were crossed with Ldlr−/− mice, and the resulting P4h-tm+/−/Ldlr+/− mice were subsequently bred to obtain P4h-tm+/−/Ldlr−/− mice, which were crossed to produce P4h-tm−/−/Ldlr−/− and P4h-tm+/+/Ldlr−/− mice (used as controls). Three-month-old males were used in all experiments. The mice were first fed a standard rodent diet (Teklad 2018, Envigo) after which they received a HFD (42% from fat, TD.88137, Envigo) for 13 weeks. Sex is a critical biological variable33; however, we did not use female mice due to results of several animal studies suggesting that estrogens have an inhibitory effect on a diet-induced atherosclerosis in mice34 and strict adherence to 3R principles.

Determination of Serum Lipids and Lipoprotein Profiles

Serum total cholesterol, HDL (high-density lipoprotein) cholesterol, triglycerides, and LDL+VLDL (very LDL) cholesterol levels were determined as described.16 Serum for lipoprotein profiling was obtained after a 12-hour night fast. Fractionation of serum was accomplished by fast protein liquid chromatography on a Superose 6 10/300 GL column (Amersham Pharmacia). Two independent fast protein liquid chromatography assays with pooled serum from 5 animals per genotype, derived from randomly chosen mice at the same age from different breeding rounds, were performed. Triglycerides and cholesterol in the lipoprotein fractions were measured as above.

Analysis of Atherosclerosis

The atherosclerosis study adhered to the guidelines for experimental atherosclerosis studies.35 The extent of atherosclerosis was analyzed using en face plaque staining of the full-length aorta with Sudan IV, as described.36 Five-micrometer sections of formaldehyde-fixed paraffin-embedded tissue samples were stained with hematoxylin-eosin and photographed with Leica DM LB2 microscope and Leica DFC 320 camera. The hearts were sectioned to localize the aortic origin, and the plaque area was quantified in 3 consecutive sections at 75 µm interval using the Photoshop software.

Hepatic Histology and Hepatic Triglycerides

Liver steatosis was quantified from hematoxylin-eosin stained sections, and the amount of collagen in the liver was determined with Masson trichrome staining as described.15,16 Hepatic lipids were extracted and measured as described.16

RNASeq

Three hundred nanograms total RNA, extracted from P4h-tm+/+/Ldlr−/− and P4h-tm−/−/Ldlr−/− liver, was used for library preparation. The libraries were sequenced on Illumina NextSeq550 platform followed by FASTQ generation within BaseSpace (Illumina). The resulting 25.38 Gbp of data were with average Q30 values of 92.72%. Analyses were performed with BaseSpace Sequence Hub with Mus musculus reference genome. After DESeq2 analyses, only significantly DE genes (P≤0.05) were incorporated. Pathway analyses were performed with ConsensusPathDB, including only KEGG pathways known in the Mus musculus database.

Analysis of Circulating Antibodies

The modified lipoprotein antigens CuOx-LDL (copper-OxLDL) and MAA-LDL (malondialdehyde-acetaldehyde-oxidized LDL) were prepared and used in chemiluminescence immunoassays to determine autoantibody binding, as described.37 The total serum immunoglobulin concentrations were determined by chemiluminescent immunoassay.38

Western Blot Analyses

Western blot analyses were performed using standard protocols with antibodies listed in Major Resources Table, and quantified with Fiji software (https://imagej.net/ImageJ_Ops).

Quantitative Real-Time PCR Analyses

RNA extraction and qPCR analyses were performed as described16 with primers shown in Table I in the Data Supplement.

Statistical Analyses

All individual data as scatterplots and means±SEM as bar graphs are presented. Prior statistical tests, normality, and equal variance analyses were performed. Statistical differences were evaluated using Student 2-tailed t test for normal distributed data. Non-normal distributed data sets were log-transformed to meet the assumption of normality before t test was conducted. P≤0.05 was considered statistically significant. Significances of RNASeq data are shown via adjusted P values in the single genes and q values in the pathways. Pearson correlation coefficient was calculated to compare linear dependences between 2 variables.

Results

Generation of a P4h-tm−/−/Ldlr−/− Double Knockout Mouse Line for Experimental Atherosclerosis

To generate an experimental model for studying the role of P4H-TM inhibition in atherogenesis, we crossed P4h-tm−/− and Ldlr−/− mice and analyzed the expression of P4H-TM mRNA and protein in the tissues of P4h-tm−/−/Ldlr−/− mice by qPCR and X-gal staining (Figure I in the Data Supplement), respectively. qPCR data revealed very low P4h-tm expression in WAT and liver and low in heart and lung, and an increased expression of truncated P4h-tm mRNA in P4h-tm−/−/Ldlr−/− mice compared with P4h-tm+/+/Ldlr−/− littermates. Increased expression of the truncated P4h-tm mRNA in various tissues of the P4h-tm−/− mice was already reported26 and suggests a feedback mechanism for the induction of P4h-tm mRNA transcription in tissues lacking the P4H-TM protein. In line with mRNA data, no X-gal staining was detected in the WAT, liver and heart of these mice, indicating low endogenous expression of P4H-TM in these tissues. The strong staining seen in the brain of the P4h-tm−/−/Ldlr−/− mice was already reported in P4h-tm−/− mice26 (Figure I in the Data Supplement). These mice together with their P4h-tm+/+/Ldlr−/− littermates were fed on HFD for 13 weeks and euthanized. The weight gain of the animals was monitored weekly during this period, and no significant differences were observed between the genotypes (Figure II in the Data Supplement). At euthanization, the average weights of the liver and spleen were 16% and 52% greater, respectively, in the P4h-tm−/−/Ldlr−/− mice than in the P4h-tm+/+/Ldlr−/− mice, although these differences were not significant. In the case of the spleen, the difference was mostly due to one very heavy spleen among the P4h-tm−/−/Ldlr−/− mice. There was no significant difference in the weight of gonadal WAT between the genotypes at euthanization (Figure II in the Data Supplement). Additionally, the expression levels of Hif-p4h-1-3 and P4h-tm mRNA of the P4h-tm+/+/Ldlr−/− and P4h-tm−/−/Ldlr−/− mice fed on HFD for 13 weeks were studied (Figure III in the Data Supplement). The mRNA levels of Hif-p4h-1 and Hif-p4h-2 in the liver, heart, and lung were not altered while a 30% decrease in the mRNA levels of Hif-p4h-3 in the liver but not in the heart and lung was detected.

Lower Serum Triglyceride Levels in the P4h-tm−/−/Ldlr−/− Mice

Analysis of the serum lipid levels after 13 weeks on HFD showed very high levels of total cholesterol, HDL cholesterol, and LDL+VLDL cholesterol in all mice, and no difference in these or in the HDL/LDL+VLDL cholesterol ratio was observed between the genotypes (Figure 1A). The P4h-tm−/−/Ldlr−/− mice had ≈40% lower serum triglyceride levels after 13 weeks on HFD as compared with P4h-tm+/+/Ldlr−/−, and a similar difference was detected at 9 weeks (Figure 1B). Interestingly, the difference between the genotypes was already detected when fed standard laboratory diet (week 0, at the age of 3 months), at which point the P4h-tm−/−/Ldlr−/− mice had ≈40% lower serum triglyceride levels than P4h-tm+/+/Ldlr−/− (Figure 1B). We, therefore, analyzed the serum lipid panel in the P4h-tm−/− mice fed standard laboratory diet, and found no difference in total cholesterol or HDL cholesterol levels between the genotypes. However, the P4h-tm−/− mice had significantly lower triglyceride levels than the P4h-tm+/+ mice (Figure 1C). While crossing of the P4h-tm+/+ mice with Ldlr−/− mice significantly increased their serum triglyceride levels (1.52±0.12 versus 2.40±0.48 mmol/L, P=0.00097), no increase in triglyceride was seen in the P4h-tm−/− versus P4h-tm−/−/Ldlr−/− mice (1.52±0.10 versus 1.30±0.10 mmol/L, P=0.35; Figure 1B and 1C). To gain a better understanding of the lower triglyceride levels in the P4h-tm−/−/Ldlr−/− mice, we analyzed their serum lipoprotein fractions for triglyceride and cholesterol. Fast protein liquid chromatography analyses showed no differences between the genotypes in the levels or distribution of triglyceride and cholesterol associated with serum lipoprotein fractions (Figure 1D). Interestingly, more free glycerol was detected in the serum lipoprotein profile analyses in the P4h-tm−/−/Ldlr−/− mice (Figure 1D).

Figure 1.

Figure 1. P4h-tm−/−/Ldlr−/− mice had reduced serum triglyceride levels.A, Serum total cholesterol, HDL (high-density lipoprotein) cholesterol, and LDL (low-density lipoprotein)+VLDL (very-LDL) cholesterol levels and HDL/LDL+VLDL cholesterol ratios in P4h-tm+/+/Ldlr−/− and P4h-tm−/−/Ldlr−/− male mice measured after 13 wk on high-fat diet (HFD). B, Serum triglyceride (TG) levels at 0, 9, and 13 wk on HFD. n=10 (P4h-tm+/+/Ldlr−/−), n=7 (P4h-tm−/−/Ldlr−/−). C, Serum total cholesterol, HDL cholesterol, and serum TG levels in P4h-tm+/+ and P4h-tm−/− male mice fed standard laboratory diet after 12 h night fasting. n=9 (P4h-tm+/+), n=12 (P4h-tm−/−). Error bars, means±SEM. *P≤0.05, **P≤0.01. D, Serum lipoprotein profiles in P4h-tm+/+/Ldlr−/− and P4h-tm−/−/Ldlr−/− mice fed on standard laboratory diet after 12 h fasting. Serum lipoprotein profiles were determined by high-resolution size-exclusion chromatography, and the fractions were analyzed for TG and total cholesterol. Each profile represents pooled sera from 5 mice.

P4H-TM Deficiency Reduced Atherosclerotic Plaques in the Full-Length Aorta

The area of atherosclerotic plaques in relation to the area of the full-length aorta when analyzed en face from the Sudan IV-stained specimens after 13 weeks on HFD showed a 32% reduction in the P4h-tm−/−/Ldlr−/− mice as compared with P4h-tm+/+/Ldlr−/− (Figure 2A and 2B). No significant difference at the aortic cross-section level, in the area of plaques relative to the area of the aortic valves, between the genotypes was detected (Figure 2C and 2D). Interestingly, the area of the full-length aorta was 18% larger in the P4h-tm−/−/Ldlr−/− mice than in the P4h-tm+/+/Ldlr−/− genotype (0.98±0.08 versus 0.83±0.04 cm2, P=0.05) and that of the aortic valves 36% larger in the former (2.01±0.25 versus 1.48±0.15 μm2, P=0.07). Although the P4h-tm−/−/Ldlr−/− mice showed a trend towards reduced area of necrotic core in the aortic plaques at the valvular level compared with P4h-tm+/+/Ldlr−/−, when analyzed as a proportion of the whole area of plaque, this difference (≈30%) was not significant (Figure 2E and 2G). No difference was seen in the amount of plaque collagen and total area of foam macrophages (Figure 2F and 2H).

Figure 2.

Figure 2. P4h-tm−/−/Ldlr−/− mice had reduced aortic plaques in the full-length aorta.A, Aortic plaques in full-length aortas of P4h-tm+/+/Ldlr−/− and P4h-tm−/−/Ldlr−/− male mice after 13 wk on high-fat diet (HFD), analyzed with Sudan IV staining. B, Ratio of plaque area to total aortic area. C, Hematoxylin-eosin stained aortic cross-section at the valvular level. Scale bar=500 µm. D, Measured plaque area in the cross-section of the aortic origin. E, Masson trichrome staining used to analyze the collagen in the aortic plaques (F) necrotic core (G) and foam macrophage areas (H). Scale bar=500 µm. n=10 (P4h-tm+/+/Ldlr−/−), n=7 (P4h-tm−/−/Ldlr−/−). Error bars, means±SEM. *P≤0.05.

Analysis of Circulating Autoantibodies to Ox-LDL Identified Higher IgM Levels in the P4h-tm−/−/Ldlr−/− Mice

CuOx-LDL and MAA-LDL, MAA being a derivative of malondialdehyde, represent model antigens are which are used to assess specific immune responses to Ox-LDL.5 The levels of the circulating autoantibodies to Ox-LDL in the sera of the P4h-tm−/− mice fed standard laboratory diet were similar to P4h-tm+/+ (Figure IV in the Data Supplement). When the levels of circulating IgG and IgM natural antibodies against CuOx-LDL or MAA-LDL, and levels of total IgG and IgM antibodies were analyzed in P4h-tm+/+/Ldlr−/− and P4h-tm−/−/Ldlr−/− mice fed standard laboratory diet (0 week) and after 9 and 13 weeks on HFD, no differences were observed between the genotypes in the levels of total IgG, CuOx-LDL IgG, or MAA-LDL IgG at any time point (Figure 3A through 3C). Similarly, there was no difference between the genotypes in the levels of total IgM, CuOx-LDL IgM, or MAA-LDL IgM at 0 week (Figure 3D through 3F). However, the levels of total IgM and those of the CuOx-LDL IgM fractions had increased significantly in the P4h-tm−/−/Ldlr−/− serum compared with P4h-tm+/+/Ldlr−/− during the HFD feeding at 9 weeks, and these differences persisted at 13 weeks (Figure 3D and 3E). No differences in the MAA-LDL IgM fractions between the genotypes were detected at 9 or 13 weeks (Figure 3F). Next, the total amount of B cells in the plaques and spleen, and the B-cell subsets in spleens after 13 weeks on HFD were studied (Figure V in the Data Supplement); however, no differences in total B cells in plaques and spleen or in splenic B1 cells, considered the major source of natural antibodies,5 between the genotypes were detected.

Figure 3.

Figure 3. The P4h-tm−/−/Ldlr−/− mice displayed increased amounts of atheroprotective IgM autoantibodies against CuOx-LDL (copper-oxidized low-density lipoprotein).A, Serum total IgG (B) IgG bound to CuOx-LDL, (C) IgG bound to MAA-LDL (malondialdehyde-acetaldehyde-modified LDL), (D) serum total IgM, (E) IgM bound to CuOx-LDL, and (F) IgM bound to MAA-LDL, all measured after 0, 9, and 13 wk on high-fat diet (HFD). n=10 (P4h-tm+/+/Ldlr−/−), n=6/7 (P4h-tm−/−/Ldlr−/−). Error bars, means±SEM. *P≤0.05. P values in E were calculated from the log-transformed values. RLU indicates relative light units.

The P4h-tm−/−/Ldlr−/− Mice Had a Trend for More Hepatic Triglycerides and Gene Expression Alterations in Pathways Regulating Lipid Metabolism and Immunologic Responses in the Liver

Histological analyses of the livers of the mice after 13 weeks on HFD revealed severe steatosis in both genotypes (Figure 4A). All the livers of the P4h-tm−/−/Ldlr−/− mice were highly steatotic, whereas the P4h-tm+/+/Ldlr−/− group included some less affected individuals. To quantify the difference, we analyzed biochemically the amount of triglyceride in the livers and found ≈20% higher levels in the P4h-tm−/−/Ldlr−/− than in the P4h-tm+/+/Ldlr−/− livers, although this difference was not significant (Figure 4B). No fibrosis was detected in either genotype (data not shown).

Figure 4.

Figure 4. P4h-tm−/−/Ldlr−/− mice had a trend for increased hepatic steatosis and gene expression alterations in pathways regulating lipid metabolism and immunologic responses in the liver.A, Scoring of the severity of steatosis from the hematoxylin-eosin stained liver sections. B, Liver triglyceride levels. n=10 (P4h-tm+/+/Ldlr−/−), n=7 (P4h-tm−/−/Ldlr−/−). Error bars, means±SEM. C–F, RNA-sequencing analysis of hepatic transcriptomes of P4h-tm−/−/Ldlr−/− and P4h-tm+/+/Ldlr−/− mice after 13 wk on high-fat diet (HFD). C, Principal component analysis of RNA expression. D, Heatmap of 442 significantly differentially expressed genes between P4h-tm+/+/Ldlr−/− and P4h-tm−/−/Ldlr−/− livers. List of selected canonical pathways upregulated (E), and downregulated (F), between the genotypes. n=2 (P4h-tm+/+/Ldlr−/−), n=2 (P4h-tm−/−/Ldlr−/−). FDR indicates false discovery rate; PPAR, peroxisome proliferator-activated receptor; and TRP channels, transient receptor potential channels.

We next analyzed hepatic gene expression in the P4h-tm+/+/Ldlr−/− and P4h-tm−/−/Ldlr−/− mice after 13 weeks on HFD by means of RNASeq. The principal component analysis indicated that the 2 genotypes differed (Figure 4C) in terms of the expression of 442 genes (Figure 4D, Table II in the Data Supplement). Pathway analysis showed increased expression of RNAs in multiple pathways in the P4h-tm−/−/Ldlr−/− versus P4h-tm+/+/Ldlr−/− livers related to immunologic and inflammatory processes such as phagosomes, FcγR-mediated phagocytosis, chemokine signaling, and antigen processing and presentation. The P4h-tm−/−/Ldlr−/− livers also expressed RNAs involved in cell adhesion and cholesterol metabolism pathways to a greater extent than did the P4h-tm+/+/Ldlr−/− livers (Figure 4E, Table III in the Data Supplement). In contrast, the pathways that involved downregulated RNAs in the P4h-tm−/−/Ldlr−/− livers as compared with P4h-tm+/+/Ldlr−/− were complement and coagulation cascades, several pathways associated with amino acid metabolism, omega-6-fatty acid pathways (arachnoid acid and linoleic acid), fatty acid degradation, steroid hormone synthesis, retinol metabolism, and peroxisome pathways (Figure 4F, Table III in the Data Supplement).

P4H-TM Deficiency Modulates LPL Protein Levels in WAT and Serum

Although our RNASeq data showed differential Lpl RNA expression between the genotypes, qPCR validation analyses revealed low expression of hepatic Lpl mRNA and no significant difference between the genotypes (Figure 5A). Likewise, there was no difference in hepatic LPL protein levels between the genotypes (Figure 5B). Instead, we found an almost significant increase in levels of WAT Lpl mRNA of the P4h-tm−/−/Ldlr−/− mice relative to P4h-tm+/+/Ldlr−/− after 13 weeks on HFD (Figure 5A). These changes were reflected on the LPL protein levels in a significant manner as P4h-tm−/−/Ldlr−/− mice showed ≈2.5-fold more LPL in WAT than the P4h-tm+/+/Ldlr−/− mice (Figure 5B). Additionally, serum LPL protein levels were significantly higher in the P4h-tm−/−/Ldlr−/− mice than in the P4h-tm+/+/Ldlr−/− genotype both on standard laboratory diet and on HFD (Figure 5C). Although LPL liberates free fatty acids, no difference in the serum FFA (free fatty acids) levels was detected between the genotypes (Figure 5D).

Figure 5.

Figure 5. P4h-tm−/−/Ldlr−/− mice had increased Lpl (lipoprotein lipase) mRNA levels in white adipose tissue (WAT) and LPL protein levels in WAT and sera.A, qPCR analysis of Lpl mRNA in the liver and WAT of P4h-tm+/+/Ldlr−/− and P4h-tm−/−/Ldlr−/− male mice after 13 wk on high-fat diet (HFD). n=10 (P4h-tm+/+/Ldlr−/−), n=7 (P4h-tm−/−/Ldlr−/−).B, Representative Western blot analyses and densitometric quantification of LPL in liver and WAT from P4h-tm+/+/Ldlr−/− and P4h-tm−/−/Ldlr−/− mice after 13 wk on HFD. Vinculin was used as a loading control. n=10 (P4h-tm+/+/Ldlr−/−), n=7 (P4h-tm−/−/Ldlr−/−). C, Western blot analyses and densitometric quantification of LPL in sera from P4h-tm+/+/Ldlr−/− and P4h-tm−/−/Ldlr−/− male mice fed on standard laboratory diet (baseline [BL]; n=4 [P4h-tm+/+/Ldlr−/−], n=4 [P4h-tm−/−/Ldlr−/−]) or on HFD after 13 wk, (n=3 [P4h-tm+/+/Ldlr−/−], n=5 [P4h-tm−/−/Ldlr−/−]). Ponceau S staining was used as a loading control. D, Serum FFA (free fatty acids) levels, n=5 (P4h-tm+/+/Ldlr−/−), n=8 (P4h-tm−/−/Ldlr−/−). Error bars, means±SEM. *P≤0.05.

Serum Triglyceride Levels Were Associated Negatively With IgM Autoantibodies Against CuOx-LDL

Serum triglyceride levels, which were significantly lower in the P4h-tm−/−/Ldlr−/− mice than in the P4h-tm+/+/Ldlr−/− mice at 13 weeks (Figure 1B), correlated positively with total cholesterol, HDL cholesterol and LDL+VLDL cholesterol levels at the same time point (Figure 6A through 6C, respectively). The percentage of plaque area to total aortic area, which was significantly lower in the P4h-tm−/−/Ldlr−/− mice (Figure 2A and 2B), showed a significant positive correlation with the area of the necrotic core (Figure 6D). In addition, there was a positive correlation between the plaque size at the aortic origin and foam macrophages (Figure 6E). Serum triglyceride levels at baseline had an almost significant negative association with serum LPL protein levels at baseline (r=−0.690, P=0.058; Figure 6F), and a similar negative trend persisted at 13 weeks, suggesting that the reduction in triglyceride levels in the sera of the P4h-tm−/−/Ldlr−/− mice may have been associated with the increased LPL protein levels. Serum triglyceride levels at 13 weeks correlated significantly negatively with CuOx-LDL IgM and MAA-LDL IgM at 13 weeks (Figure 6J and 6K), the CuOx-LDL IgM levels being significantly higher in the P4h-tm−/−/Ldlr−/− sera compared with P4h-tm+/+/Ldlr−/− (Figure 3E). In addition, we performed principal component analysis and built a predictive logistic regression model on multiple variable data including s-triglyceride, CuOx-IgM, total IgM, and LPL in WAT (Figure VI in the Data Supplement). Principal component analysis suggested a strong relationship between s-triglyceride and IgMs, while LPL appeared more distinct. The obtained AUC=0.814 for the logistic regression model represents a measure of the model accuracy and implied that we could distinguish between P4h-tm+/+/Ldlr−/− and P4h-tm−/−/Ldlr−/− phenotype based on these variables with good confidence.

Figure 6.

Figure 6. Correlation analyses of the parameters studied in relation to atherosclerosis. Correlations between serum triglyceride (TG) levels and (A) serum cholesterol levels at 13 wk, (B) serum HDL (high-density lipoprotein) cholesterol levels at 13 wk, and (C) serum LDL (low-density lipoprotein)+VLDL (very LDL) cholesterol levels at 13 wk. Correlations between (D) the histologically quantified necrotic core of the aortic cross-section and the ratio of plaque area to total aortic area and (E) the foam macrophages of the aortic cross-section and the aortic cross-section at the valvular level. F, Correlations between the levels of serum TG and LPL (lipoprotein lipase) protein in sera at BL (baseline, mice fed on standard laboratory diet). Correlations between (G) LPL protein levels and serum TG at 13 wk, (H) CuOx-LDL (copper-oxidized LDL) IgG and serum TG levels at 13 wk, and (I) MAA-LDL (malondialdehyde-acetaldehyde-modified LDL) IgG and serum TG levels at 13 wk. Correlations between (J) CuOx-LDL IgM and serum TG levels at 13 wk and (K) MAA-LDL IgM and serum TG levels at 13 wk. *P≤0.05 and **P≤0.01.

Discussion

Nothing was known about the potential contribution of the assumed fourth HIF-P4H, P4H-TM to atherosclerosis before this study. Data on the other HIF-P4H isoenzymes suggest that their inhibition offers atheroprotection via metabolic, inflammatory, and immunologic mechanisms.15,21,23 On the contrary, activation of the hypoxia response/HIF1, especially in plaques, has been linked to the opposite findings, with increased atherosclerosis and an atheromatous inflammatory plaque phenotype.39,40P4h-tm−/−/Ldlr−/− mice have no changes in the gene expression levels of Hif-p4h-1 and Hif-p4h-2 that may suggest a compensatory effect; however, a decreased hepatic Hif-p4h-3 mRNA was detected. Hepatic Hif-p4h-3 deficiency was reported to improve the glucose and lipid metabolism via stabilization of HIF2α41 whereas in this study a severe steatosis and a trend for increased liver triglyceride in the P4h-tm−/−/Ldlr−/− mice was detected. Thus, most likely the decreased hepatic Hif-p4h-3 mRNA did not account for the observed atheroprotective phenotype in the P4h-tm−/−/Ldlr−/− mice. However, we cannot exclude completely this possibility since the hepatic HIF-P4H-3 protein levels/function and its reintroduction were not assessed in this study.

High triglyceride levels have been identified as an independent risk factor for atherosclerosis,42,43 and it has been speculated that further pharmacological approaches in addition to statins might be beneficial with regard to the prognosis for high-risk patients (cases with diabetes or metabolic syndrome) presenting with increased serum triglyceride levels. Our P4h-tm−/−/Ldlr−/− mice had significantly reduced serum triglyceride levels compared with their P4h-tm+/+/Ldlr−/− counterparts before the introduction of HFD, and their levels remained lower after 13 weeks on HFD. There was no difference in serum total cholesterol, HDL, or LDL+VLDL cholesterol levels between the genotypes, although there was a positive correlation between serum triglyceride levels and cholesterol after 13 weeks on HFD.

Depending on their source of origin and release, triglycerides are circulating in the blood packed in VLDL (endogenous pathway, liver) or in chylomicrons (exogenous pathway, intestine).44 Their concentration in blood at any given time represents the balance between the rates of entry and removal. Both VLDL- and chylomicron-derived triglycerides are hydrolyzed by LPL providing free fatty acids and glycerol for tissue use and leading to the formation of atherogenic remnant particles.44 Thus, plasma triglyceride reflects remnant particles, which include VLDL, intermediate-density lipoproteins, and in the nonfasting state chylomicron remnants. Like LDL cholesterol, both VLDL and chylomicrons can be ingested by macrophages into the arterial wall and can increase inflammation and plaque growth, resulting in atherosclerosis, but unlike LDL, they do not require oxidative modifications for the promotion of atherogenesis.45 We did not detect a difference in LDL+VLDL levels between the genotypes and the reduction in serum triglyceride levels in P4h-tm−/−/Ldlr−/− mice was not reflected in the serum lipoprotein profiles. Considering that increased VLDL levels are the default lipoprotein disturbance in mild-to-moderate hypertriglyceridemia, after which the effect of chylomicrons increases in importance,46 the lower serum triglyceride levels in P4h-tm−/−/Ldlr−/− mice could commence with decreased chylomicron levels in their serum. The lower triglyceride levels may thus have originated from reduced triglyceride release from the intestinal cells of P4h-tm−/−/Ldlr−/− mice, which was not assessed here. With that in mind, the detected higher LPL levels in the P4h-tm−/−/Ldlr−/− sera, which likely originated from the adipose tissue, may be a compensatory mechanism for reduced triglyceride release from the intestine. This is further supported by the higher free glycerol levels in P4h-tm−/−/Ldlr−/− serum compared with P4h-tm+/+/Ldlr−/− which may be a reflection on an increased rate of lipolysis despite no difference in FFA, given the faster turnover of FFA and lower metabolic utilization of glycerol. Our findings clearly indicate that the lower serum triglyceride levels in the P4h-tm−/−/Ldlr−/− mice were associated with P4H-TM deficiency, since analysis of a cohort of P4h-tm+/+ and P4h-tm−/− mice showed significantly less serum triglyceride in the P4h-tm−/− mice. Since P4H-TM is highly expressed in the brain,26,27 it is tempting to speculate that a brain-gut axis could contribute to the regulation of the triglyceride homeostasis. Thus, P4H-TM deficiency in the brain, via yet unknown neuronal/hormonal network, may have reduced the chylomicron output.

Immunology has been established as a core modulator of atherosclerosis,3,4 and IgM autoantibodies against modified LDL particles have, in particular, been shown to grant protection from plaque accumulation and development.5 The P4h-tm−/−/Ldlr−/− mice had significantly higher levels of total IgM and CuOx-LDL IgM at 9 and 13 weeks on HFD as compared with the P4h-tm+/+/Ldlr−/− mice. Interestingly, the levels of CuOx-LDL IgM correlated negatively with serum triglyceride levels, this being in agreement with the atheroprotection observed in the P4h-tm−/−/Ldlr−/− mice that presented with higher CuOx-LDL IgM levels and lower serum triglyceride levels than the P4h-tm+/+/Ldlr−/− mice. These data suggest that inhibition of P4H-TM combined with HFD feeding may be a means for promoting innate immune responses that can mediate protection of these mice from atherosclerosis.

The RNASeq data showed an increase in RNAs in the phagosome, FcγR-mediated phagocytosis, cell adhesion, and antigen processing and presentation pathways in P4h-tm−/−/Ldlr−/− as compared with P4h-tm+/+/Ldlr−/− livers, suggesting an active immunologic response in the liver. This does not fully translate to processes involved in the plaques; however, hepatic inflammation has been associated with the development of atherosclerosis as an inflammatory entity.47 Furthermore, our RNASeq data showed a difference in hepatic expression of Lpl RNA between the genotypes. LPL is a key enzyme responsible for hydrolyzing triglycerides in chylomicrons and VLDL and plays a critical role in regulating lipid metabolism and transport.44 Our qPCR validation analyses revealed low expression of hepatic Lpl mRNA and no difference between the genotypes. Instead, we detected almost significantly higher levels of Lpl mRNA in P4h-tm−/−/Ldlr−/− WAT, which is among the primary tissues for the synthesis of LPL. Likewise, the LPL protein levels were significantly upregulated in the WAT of P4h-tm−/−/Ldlr−/− mice by comparison with the P4h-tm+/+/Ldlr−/− mice.

Overexpression of LPL in a Ldlr−/− background has been reported to make the mice resistant to diet-induced atherosclerosis, mainly due to the suppression of remnant lipoproteins.48 LPL overexpression in the ApoE−/− background resulted in a similar phenotype to that seen in P4h-tm−/−/Ldlr−/− mice with decreased serum triglyceride levels and less aortic lesions, with no differences in the levels of serum total cholesterol and HDL.49 Moreover, the atheroprotective role of plasma LPL is well established, even though some controversial results have been reported.50 Furthermore, our data showed increased LPL protein levels in WAT of P4h-tm−/−/Ldlr−/− mice after 13 weeks on a HFD and in sera from both before and after exposure to HFD, and also a negative correlation between LPL protein levels and triglyceride in sera. Although, serum LPL is considered to be catalytically inactive,51 its amount in serum has been shown to be positively associated with its systemic biosynthesis and ligand function in both mice and humans.52 Moreover, the amount of serum LPL has been reported to be significantly associated with plasma triglyceride or HDL cholesterol levels53,54 and to be more closely correlated with triglyceride and LPL-mediated lipoprotein metabolism than postheparin LPL.52 In humans, decreased or deficient LPL activity resulted in hypertriglyceridemia associated with insulin resistance and type II diabetes, which in turn are factors that contribute to the development of atherosclerosis.55 Also, patients with coronary atherosclerosis have been reported to have considerably lower levels of serum LPL mass than healthy controls.56

All in all, the P4h-tm−/−/Ldlr−/− mice appeared to be protected from atherosclerosis due to their immuno-metabolic phenotype characterized with reduced serum triglyceride levels, increased LPL levels in WAT and sera, and promoted innate immune responses. Although, the exact mechanisms remain to be determined, our results establish P4H-TM inhibition as a novel potential treatment strategy for providing protection against the development of atherosclerosis.

Nonstandard Abbreviations and Acronyms

CuOx-LDL

copper-oxidized LDL

HDL

high-density lipoprotein

HFD

high-fat diet

HIF

hypoxia-inducible factor

HIF-P4Hs

hypoxia-inducible factor prolyl 4-hydroxylases

LDL

low-density lipoprotein

LPL

lipoprotein lipase

MAA-LDL

malondialdehyde-acetaldehyde-modified low-density lipoprotein

Ox-LDL

oxidized LDL

P4H-TM

transmembrane prolyl 4-hydroxylase

VLDL

very low-density lipoprotein

WAT

white adipose tissue

Acknowledgments

We thank T. Aatsinki, E. Lehtimäki, and S. Rannikko for their excellent technical assistance, Biocenter Oulu Sequencing Center for the RNASeq analyses, the Transgenic Core Facility of Biocenter Oulu and the Oulu Laboratory Animal Centre Research Infrastructure, University of Oulu, Finland, for the support.

Disclosures J. Myllyharju owns equity in FibroGen Inc, which develops HIF-P4H inhibitors as potential therapeutics. The company supports research in her laboratory. The other authors report no conflicts.

Footnotes

*E.Y. Dimova and P. Koivunen are joint senior authors.

The Data Supplement is available with this article at https://www.ahajournals.org/doi/suppl/10.1161/ATVBAHA.121.316034.

For Sources of Funding and Disclosures, see page 2139.

Correspondence to: Peppi Koivunen, MD, PhD, Faculty of Biochemistry and Molecular Medicine, University of Oulu, Aapistie 7C, FIN-90014 Oulu, Finland, Email

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