Lysophospholipids, Lysophosphatidic Acids and Monoacylglycerols: New Therapeutic Targets in Cardiovascular Diseases?

Cardiovascular diseases (CVD) are the leading cause of premature death and disability in humans. Increasing data suggest that CVD is closely related to lipid metabolism and signaling. This study aimed to assess whether circulating lysophospholipids (LPL), lysophosphatidic acids (LPA) and monoacylglycerols (MAG) may be considered as biomarkers of CVD. For this objective, the evolution of the plasma levels of 22 compounds (13 LPL, 6 LPA and 3 MAG) was monitored by liquid chromatography coupled with tandem mass spectrometry (HPLC/MS²) in different rat models of CVD, i.e. angiotensin-II-induced hypertension (HTN), ischemic chronic heart failure (CHF) and sugen/hypoxia(SuHx)-induced pulmonary hypertension (PH). On one hand, there was modest changes on the monitored compounds in HTN (LPA 16:0, 18:1 and 20:4), LPC 16:1) and CHF (LPA 16:0, LPC 18:1 and LPE 16:0 and 18:0) models compared to control rats but these changes were no longer significant after correction for multiple testing. On the other hand, PH was associated with important changes in plasma LPA with a significant increase in the 16:0, 18:1, 18:2, 20:4 and 22:6 species. A deleterious impact of LPA was confirmed on isolated human pulmonary smooth muscle cells with an increase in their proliferation. This study demonstrates that circulating LPA species are increased in rats with PH and may contribute to the pathophysiology of this disease. Additional experiments are needed to assess whether the modulation of LPA signaling in PH may be of interest.


Introduction
Cardiovascular diseases (CVD) are the leading cause of mortality and a major contributor to disability. In 2019, 523 million cases and 18.6 millions of deaths were reported corresponding to a 100% and a 50% increase since 1990 respectively. Because CVD remain the leading cause of disease burden in the world and still rise in all countries, the need to phosphatidic acid acyltransferases, LPL: lysophospholipids, LPP: lysophosphatidic acid phosphatase, lysoPL: lysophospholipase, MAG: monoacylglycerol, MAGK: monoacylglycerol kinase, PA: phosphatidic acid, PLA: phospholipase Thus, this study aims to evaluate the evolution of circulating LPL, LPA and MAG levels in different rat models of CVD, i.e., hypertension (HTN), heart failure (HF) and pulmonary hypertension (PH) in order to provide new insights to the interest of targeting LPA metabolism with pharmacological compounds.

Impact of LPA on pulmonary artery smooth muscle cell (PA-SMC) proliferation
To better evaluate whether LPA species may be involved in the pathophysiology of PH, we investigated their impact on the proliferation of human pulmonary artery smooth muscle cells (SMC) assessed using 5-bromo-2-deoxyuridine (BrdU) incorporation. Of interest, the conducted ANOVA was significant (p=0.004) and all tested LPA species (LPA(18:1), LPA(18:2) and LPA(20:4)) induced an increase SMC proliferation ( Figure 6).

Discussion
The major finding of the present work is that amongst the three well-characterized models of CVD, induction of PH by SuHx strongly increased plasma LPA and that these lipid mediators potentiated the proliferation of isolated human PA-SMC.

LPL, LPA and MAG quantitation analytical method
The present analytical method aimed to monitor 22 compounds. The choice has been made to investigate compounds with and without analytical standards to have the widest possible overview of LPA metabolism based on known fragmentation patterns for each class. This leads to the limit that results are expressed as the compound-to-17:1-LPA area under the curve ratio. Furthermore, since we did not have specific internal standard for each class, we cannot be sure of the reliability of the correction of matrix effects for LPL and MAG. However, for each model, all samples were prepared and analyzed uninterruptedly resulting in the absence of inter-batch variability [20]. Furthermore, the use of 17:1-LPA, even if not being the best-suited internal standard for all compounds, allowed to decrease within-batch variability normalizing area under the curve of the monitored compounds. Finally, the low dispersibility of the intra-group values allows us to be sufficiently confident about the robustness and reliability of the results obtained.
Precautions should be taken in the preparation and analysis of these compounds. First of all, since LPA are highly acidic compounds, it is strongly recommended to perform an acidic extraction since extractions at non-acidic pH will result in poor recovery. This could be done using hydrochloric or formic acid. Here, we used formic acid to avoid production of LPA by LPL hydrolysis that could be induced by too acidic solutions. Furthermore, it is mandatory to chromatographically resolved LPA and LPL with the same esterified fatty acid since collision-induced dissociation may artificially produce LPA. The natural abundance of LPA being a tiny fraction of LPL, hydrolysis of only a minor part of LPL to LPA, either during extraction of analysis by the MS, could dramatically increase the concentration of these compounds [21].

CVD models
All evaluated models have been well characterized with an increase in SBP, a decrease in LVFS and an increase in mPAP for the HTN, CHF and PH model respectively. As observed in humans, the HTN and PH models were also associated with the development of LV hypertrophy and dysfunction.
The development of HTN induced a significant increase of 3 LPA species under unadjusted assumption. This may be justified by the fact that Ang-II modulates LPA1 receptor function [22] and induces LPA production by phospholipases activation as well as the production of phosphatidic acids [23][24].
CHF rats exhibited an increase in LPA (16:0) and LPE(16:0-, 18:0-) associated with a decrease in LPC(18:1). Interestingly, all increased compounds possess an esterified saturated fatty acid (SFA) suggesting a link between CHF-mediated oxidative stress and desaturase activity [25][26]. Furthermore, it is now well known that reducing intake of saturated fatty acids may reduce LDL-c and CVD risks [27][28]. SFA possessed numerous deleterious effects such as inflammation, apoptosis, mitochondrial dysfunction and oxidative stress [29] but their impact has mainly been evaluated as non-esterified fatty acids [30]. Thus, the role of esterified SFAin LPA and LPL esterified needs further investigations.
In the SuHx rat model of severe PH, LPA metabolism and activity seem to be a promising pathway to target. Indeed, 80% of the monitored LPA exhibited a 2-to 3-fold increase when compared with the control group. Furthermore, addition of LPA species in a cell media of isolated human PA-SMCs at a concentration of 1µM, which is relevant when compared with circulating LPA levels [31], promoted an increased proliferation. In fact, accumulation of PA-SMCs within the pulmonary arterial walls are one of the most prominent features of PH, which can lead to the narrowing or occlusion of pulmonary vessels and therefore plays an important role in the occurrence and development of PH [32][33][34][35]. At this time, the role of LPA remains unclear even if their increase in hypoxic pulmonary vascular remodeling has already been described [36]. Of note, LPA are able to regulate the hypoxia-inducible factor 1α (HIF-1α) [37], that can interact with enzymes and other transcription factors in order to control vascularization and tissue growth in response to hypoxic conditions [38].

LPA signaling
Since LPA metabolism is complex with distinct anabolic and catabolic pathways, it may be easier to specifically act on LPA receptors rather than try to modulate enzymes involved in LPA metabolism regulation. However, it is now well admitted that LPA activates at least 6 specific G protein-coupled receptor named as LPAR1-6. The downstream signals derived from activation of LPAR involve Rho, phospholipase C, phosphatidylinositol 3-kinase and adenylate cyclase intracellular pathways, thus producing diverse physio(patho)logical effects [18]. Recently, LPAR4 was found to contribute to elevation of blood pressure with the more potent effect for LPA 18:1 and 20:4 species [39]. Interestingly, LPAR activation exhibited different reactivity according to the acyl chain (carbon length and number of insaturations) and the esterification position (sn-1 or sn-2) of the LPA [40]. Another key point is related to the relative expression of those receptors in lung tissue where PH takes place. All LPAR1-6 were expressed in lung tissue according to https://www.genecards.org/ and to date, it has been demonstrated that LPAR1 and LPAR2 knock-down protects against pulmonary remodeling, lung inflammation and lung injury [41][42][43].

Animals
All the animal care and procedures were approved by French Animal Experimentation Ethics Committees and performed in accordance with the guidelines from the French National Research Council for the Care and Use of Laboratory Animals (Permit Numbers: Apafis #24107 approved on February 12, 2020 and #11484 approved on April 6, 2018). All experiments were performed in 10-weeks-old male wild-type Sprague-Dawley (SD/Crl) (Charles-River) rats.

Angiotensin-II induced hypertension (HTN)
Systemic arterial hypertension was induced using 4-week ang-II infusion with osmotic pumps (0.25 µg/kg/day), implanted subcutaneously in isoflurane-anesthetized rats. Non-implanted rats served as controls. Tail-cuff plethysmography was used in trained conscious animals to confirm the development of systemic hypertension.

Chronic heart failure (CHF) induced by coronary artery ligation
For ischemic HF, myocardial infarction was induced by definitive left coronary artery ligation as previously described (J Mol Cell Cardiol. 2012 Mar;52(3):660-6.). Briefly, rats were anesthetized using methohexithal (50 mg/kg i.p.), intubated and ventilated at 60 cycles/min (tidal volume 1 ml/100 g of body weight). A left thoracotomy was performed, and the heart exposed. A 6/0 polypropylene suture was passed around the proximal left coronary artery, which was tied in order to induce myocardial ischemia. Ten minutes after coronary artery ligation, the chest was closed, the pneumothorax was evacuated, and the animals were allowed to recover. Sham-operated rats, subjected to the same protocol except that the coronary artery was not occluded, served as controls. Twelve weeks after surgery, the development of HF was confirmed using transthoracic echocardiography performed in rats anesthetized with isoflurane, using a Vivid 7 ultrasound echograph (GE Healthcare, Buc, France). A two-dimensional short axis view of the left ventricle was obtained at the level of the papillary muscle, in order to record M-mode tracings. Left ventricular end-diastolic (LVEDD) and systolic diameters (LVESD), allowing the determination of LV fractional shortening (FS) as FS (%)=((LVEDD-LVESD)/LVEDD)×100.

Pulmonary hypertension (PH) induced by sugen hypoxia (SuHx)
For PH, the SuHx model, in which occlusive neointimal lesions are observed, was used as previously described [33]. Briefly, rats were injected subcutaneously with SU5416 (a VEGF-receptor antagonist; 20 mg/kg) and exposed to hypoxia (10% FiO2) for 3 weeks. Then, these rats returned to normoxia (21% FiO2) for additional 5 weeks before evaluation. Control rats were not injected and remained under normoxia for 8 weeks.
To confirm the development of PH, right ventricular hemodynamic measurements were performed in anesthetized rats using a polyvinyl catheter introduced into the right external jugular vein, advanced in the right ventricle, and further, in the pulmonary artery, allowing the measurement of mean pulmonary arterial pressure.

Human PA-SMC proliferation
Lung specimens were obtained during lobectomy or pneumonectomy for localized lung cancer. The lung specimens were collected at a distance from the tumor foci. This study was approved by the local ethics committee (CPP Est-III: N°ID RCB: 2018-A01252 At the time of sacrifice, blood samples were drawn in the aorta of rats anesthetized with isoflurane using 2-mL syringes. Blood was transferred on a prechilled ethylenediaminetetraacetic acid (EDTA) tubes and immediately centrifuged 5 min at 4500g (4°C). Then, the plasma was frozen in liquid nitrogen and stored at -80°C until analysis.

Tandem mass spectrometry (MS²)
MS² was performed using a 4500QTRAP operating in the positive/negative electrospray ionization (ESI) switching mode (Sciex, Toronto, Canada). Instrument control and data acquisition were performed with Analyst 1.6.3 software. The source parameters were optimized as follow; ion spray voltage: -4500V and +4500V for negative and positive ionization mode respectively; nebulisation gas: 60 psi; desolvatation gas: 50 psi; curtain gas: 30 psi; source temperature: 500°C, entrance potential: -10V and +10V for negative and positive ionization mode respectively, collision activation dissociation: medium.  (Table 4).

Data and statistical analysis
Data, statistical analysis and captions were performed using R v4.1.0 software [44] and DescTools package [45]. For relative quantitation of LPL, data were expressed as median (interquartile range or IQR) and p-values were computed by t.test followed by Benjamini & Hochberg correction [19]. Captions were performed using ggplot2, ggsci and ggpubr packages [46][47][48]. For cell culture data analysis, ANOVA was conducted first then p-values were computed using Dunnett's post-hoc test. For LPL, LPA and MAG quantitation, due to the lack of analytical standard for several compounds, 17:1-LPA served as internal standard (IS) and data are expressed as compound-to-IS area under the curve (AUC) ratio which is unit-free. R code for statistical analysis, rat models' data and cell culture data are available in Supplementary File S1, Supplementary File S2 and Supplementary File S3 respectively.