A genetic mouse model of lean-NAFLD unveils sexual dimorphism in the liver-heart axis.

Lean patients with NAFLD may develop cardiac complications independently of pre-existent metabolic disruptions and comorbidities. To address the underlying mechanisms independent of the development of obesity, we used a murine model of hepatic mitochondrial deficiency. The liver-heart axis was studied as these mice develop microvesicular steatosis without obesity. Our results unveil a sex-dependent phenotypic remodeling beyond liver damage. Males, more than females, show fasting hypoglycemia and increased insulin sensitivity. They exhibit diastolic dysfunction, remodeling of the circulating lipoproteins and cardiac lipidome. Conversely, females do not manifest cardiac dysfunction but exhibit cardiometabolic impairments supported by impaired mitochondrial integrity and ß-oxidation, remodeling of circulating lipoproteins and intracardiac accumulation of deleterious triglycerides. This study underscores metabolic defects in the liver resulting in significant sex-dependent cardiac abnormalities independent of obesity. This experimental model may prove useful to better understand the sex-related variability, notably in the heart, involved in the progression of lean-NAFLD.

PCSK7: A novel regulator of apolipoprotein B and a potential target against non-alcoholic fatty liver disease.

BACKGROUND: Epidemiological evidence links the proprotein convertase subtilisin/kexin 7 (PCSK7) to triglyceride (TG) metabolism. We associated the known PCSK7 gain-of-function non-coding SNP rs236918 with higher levels of plasma apolipoprotein B (apoB) and the loss-of-function coding variant p.Pro777Leu (SNP rs201598301) with lower apoB and TG. Herein, we aimed to unravel the in vivo role of liver PCSK7. METHODS: We biochemically defined the functional role of PCSK7 in lipid metabolism using hepatic cell lines and Pcsk7-/- mice. Our findings were validated following subcutaneous administration of hepatocyte-targeted N-acetylgalactosamine (GalNAc)-antisense oligonucleotides (ASOs) against Pcsk7. RESULTS: Independent of its proteolytic activity, membrane-bound PCSK7 binds apoB100 in the endoplasmic reticulum and enhances its secretion. Mechanistically, the loss of PCSK7/Pcsk7 leads to apoB100 degradation, triggering an unfolded protein response, autophagy, and ß-oxidation, eventually reducing lipid accumulation in hepatocytes. Non-alcoholic fatty liver disease (NAFLD) was induced by a 12-week high fat/fructose/cholesterol diet in wild type (WT) and Pcsk7-/- mice that were then allowed to recover on a 4-week control diet. Pcsk7-/- mice recovered more effectively than WT mice from all NAFLD-related liver phenotypes. Finally, subcutaneous administration of GalNAc-ASOs targeting hepatic Pcsk7 to WT mice validated the above results. CONCLUSIONS: Our data reveal hepatic PCSK7 as one of the major regulators of apoB, and its absence reduces apoB secretion from hepatocytes favoring its ubiquitination and degradation by the proteasome. This results in a cascade of events, eventually reducing hepatic lipid accumulation, thus supporting the notion of silencing PCSK7 mRNA in hepatocytes for targeting NAFLD.

Gene-metabolite annotation with shortest reactional distance enhances metabolite genome-wide association studies results.

Metabolite genome-wide association studies (mGWAS) have advanced our understanding of the genetic control of metabolite levels. However, interpreting these associations remains challenging due to a lack of tools to annotate gene-metabolite pairs beyond the use of conservative statistical significance threshold. Here, we introduce the shortest reactional distance (SRD) metric, drawing from the comprehensive KEGG database, to enhance the biological interpretation of mGWAS results. We applied this approach to three independent mGWAS, including a case study on sickle cell disease patients. Our analysis reveals an enrichment of small SRD values in reported mGWAS pairs, with SRD values significantly correlating with mGWAS p values, even beyond the standard conservative thresholds. We demonstrate the utility of SRD annotation in identifying potential false negatives and inaccuracies within current metabolic pathway databases. Our findings highlight the SRD metric as an objective, quantitative and easy-to-compute annotation for gene-metabolite pairs, suitable to integrate statistical evidence to biological networks.

Extracellular vesicles in venous thromboembolism and pulmonary hypertension.

Venous thromboembolism (VTE) is a multifactorial disease, and pulmonary hypertension (PH) is a serious condition characterized by pulmonary vascular remodeling leading with increased pulmonary vascular resistance, ultimately leading to right heart failure and death. Although VTE and PH have distinct primary etiologies, they share some pathophysiologic similarities such as dysfunctional vasculature and thrombosis. In both conditions there is solid evidence that EVs derived from a variety of cell types including platelets, monocytes, endothelial cells and smooth muscle cells contribute to vascular endothelial dysfunction, inflammation, thrombosis, cellular activation and communications. However, the roles and importance of EVs substantially differ between studies depending on experimental conditions and parent cell origins of EVs that modify the nature of their cargo. Numerous studies have confirmed that EVs contribute to the pathophysiology of VTE and PH and increased levels of various EVs in relation with the severity of VTE and PH, confirming its potential pathophysiological role and its utility as a biomarker of disease severity and as potential therapeutic targets.

Remodeling of lipid landscape in high fat fed very-long chain acyl-CoA dehydrogenase null mice favors pro-arrhythmic polyunsaturated fatty acids and their downstream metabolites.

Very-long chain acyl-CoA dehydrogenase (VLCAD) catalyzes the initial step of mitochondrial long chain (LC) fatty acid ß-oxidation (FAO). Inherited VLCAD deficiency (VLCADD) predisposes to neonatal arrhythmias whose pathophysiology is still not understood. We hypothesized that VLCADD results in global disruption of cardiac complex lipid homeostasis, which may set conditions predisposing to arrhythmia. To test this, we assessed the cardiac lipidome and related molecular markers in seven-month-old VLCAD-/- mice, which mimic to some extent the human cardiac phenotype. Mice were sacrificed in the fed or fasted state after receiving for two weeks a chow or a high-fat diet (HFD), the latter condition being known to worsen symptoms in human VLCADD. Compared to their littermate counterparts, HFD/fasted VLCAD-/- mouse hearts displayed the following lipid alterations: (1) Lower LC, but higher VLC-acylcarnitines accumulation, (2) higher levels of arachidonic acid (AA) and lower docosahexaenoic acid (DHA) contents in glycerophospholipids (GPLs), as well as (3) corresponding changes in pro-arrhythmogenic AA-derived isoprostanes and thromboxane B2 (higher), and anti-arrythmogenic DHA-derived neuroprostanes (lower). These changes were associated with remodeling in the expression of gene or protein markers of (1) GPLs remodeling: higher calcium-dependent phospholipase A2 and lysophosphatidylcholine-acyltransferase 2, (2) calcium handling perturbations, and (3) endoplasmic reticulum stress. Altogether, these results highlight global lipid dyshomeostasis beyond FAO in VLCAD-/- mouse hearts, which may set conditions predisposing the hearts to calcium mishandling and endoplasmic reticulum stress and thereby may contribute to the pathogenesis of arrhythmias in VLCADD in mice as well as in humans.

Co-profiling reveals distinct patterns of genomic chromatin accessibility and gene expression in pulmonary hypertension caused by chronic hypoxia.

INTRODUCTION: Aberrant gene expression is a key mechanism underlying pulmonary hypertension (PH) development. The alterations of genomic chromatin accessibility and their relationship with the aberrant gene expressions in PH are poorly understood. We used bulk Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) and RNA sequencing (RNA-seq) in pulmonary artery smooth muscle cells (PASMCs) of chronic hypoxia-exposed rats mimicking group 3 human PH. METHODS: Adult Sprague Dawley rats were commercially obtained from Hunan SJA (Hunan SJA Laboratory Animal Co., Changsha, China) and randomizedly allocated into four groups exposing to nomobaric hypoxia or normoxia for 1 or 28 days respectively. After the assessment of pulmonary hemodynamics, smooth muscle cells were isolated from intralobular arteries and simultaneously subjected to bulk Assay of ATAC-seq and RNA-seq. RESULTS: Hypoxic exposure for continuous 28-days, but not for 1-day, induced established PH phenotypes in rats. ATAC-seq revealed a major distribution of differential accessibility regions (DARs) annotated to the genome in out-of-promoter regions, following 1-day or 28-days hypoxia. 1188 DAR-associated genes and 378 differentially expressed genes (DEGs) were identified in rats after exposure to 1-day hypoxia, while 238 DAR-associated genes and 452 DEGs for 28-days hypoxia. Most of the DAR-associated genes or DEGs in 1-day did not overlap with that of 28-days hypoxia. A Pearson correlation analysis indicated no significant correlation between ATAC-seq and RNA-seq. CONCLUSIONS: The alterations in genomic chromatin accessibility and genes expression of PASMCs in the initial stage of hypoxia are distinct from the established stage of hypoxia-induced PH. The genomic differential accessibility regions may not be the main mechanisms directly underlying the differentially expressed genes observed either in the initial or established stages of PH. Thus the time-course alterations of gene expression and their possible indirect link with genomic chromatin accessibility warrant more attention in mechanistic study of pulmonary hypertension.

Gene-metabolite annotation with shortest reactional distance enhances metabolite genome-wide association studies results.

Studies combining metabolomics and genetics, known as metabolite genome-wide association studies (mGWAS), have provided valuable insights into our understanding of the genetic control of metabolite levels. However, the biological interpretation of these associations remains challenging due to a lack of existing tools to annotate mGWAS gene-metabolite pairs beyond the use of conservative statistical significance threshold. Here, we computed the shortest reactional distance (SRD) based on the curated knowledge of the KEGG database to explore its utility in enhancing the biological interpretation of results from three independent mGWAS, including a case study on sickle cell disease patients. Results show that, in reported mGWAS pairs, there is an excess of small SRD values and that SRD values and p-values significantly correlate, even beyond the standard conservative thresholds. The added-value of SRD annotation is shown for identification of potential false negative hits, exemplified by the finding of gene-metabolite associations with SRD ≤1 that did not reach standard genome-wide significance cut-off. The wider use of this statistic as an mGWAS annotation would prevent the exclusion of biologically relevant associations and can also identify errors or gaps in current metabolic pathway databases. Our findings highlight the SRD metric as an objective, quantitative and easy-to-compute annotation for gene-metabolite pairs that can be used to integrate statistical evidence to biological networks.

Cryo-EM structure of the Agrobacterium tumefaciens T-pilus reveals the importance of positive charges in the lumen.

Agrobacterium tumefaciens is a natural genetic engineer that transfers DNA into plants, which is the most applied process for generation of genetically modified plants. DNA transfer is mediated by a type IV secretion system in the cell envelope and extracellular T-pili. We here report the cryo-electron microscopic structures of the T-pilus at 3.2-Å resolution and of the plasmid pKM101-determined N-pilus at 3-Å resolution. Both pili contain a main pilus protein (VirB2 in A. tumefaciens, TraM in pKM101) and phospholipids arranged in a five-start helical assembly. They contain positively charged amino acids in the lumen, and the lipids are positively charged in the T-pilus (phosphatidylcholine) conferring overall positive charge. Mutagenesis of the lumen-exposed Arg91 in VirB2 results in protein destabilization and loss of pilus formation. Our results reveal that different phospholipids can be incorporated into type IV secretion pili and that the charge of the lumen may be of functional importance.

Fatty acids derived from the probiotic Lacticaseibacillus rhamnosus HA-114 suppress age-dependent neurodegeneration.

The human microbiota is believed to influence health. Microbiome dysbiosis may be linked to neurological conditions like Alzheimer's disease, amyotrophic lateral sclerosis, and Huntington's disease. We report the ability of a probiotic bacterial strain in halting neurodegeneration phenotypes. We show that Lacticaseibacillus rhamnosus HA-114 is neuroprotective in C. elegans models of amyotrophic lateral sclerosis and Huntington's disease. Our results show that neuroprotection from L. rhamnosus HA-114 is unique from other L. rhamnosus strains and resides in its fatty acid content. Neuroprotection by L. rhamnosus HA-114 requires acdh-1/ACADSB, kat-1/ACAT1 and elo-6/ELOVL3/6, which are associated with fatty acid metabolism and mitochondrial ß-oxidation. Our data suggest that disrupted lipid metabolism contributes to neurodegeneration and that dietary intervention with L. rhamnosus HA-114 restores lipid homeostasis and energy balance through mitochondrial ß-oxidation. Our findings encourage the exploration of L. rhamnosus HA-114 derived interventions to modify the progression of neurodegenerative diseases.

MiR-1249 on Endothelial Extracellular Vesicles Mediates Cigarette Smoke-Induced Pulmonary Hypertension by Inhibiting HDAC10 (Histone Deacetylase 10)-NFκB (Nuclear Factor κB)-CaSR (Calcium-Sensing Receptor) Cascade.

BACKGROUND: Overproduction of endothelial extracellular vesicles (eEVs) is correlated with pulmonary hypertension progression, but the precise mechanism remains largely unclear. METHODS: MicroRNA-chip and real-time polymerase chain reaction were conducted to screen and validate microRNA profiles in blood plasma eEVs of rats and human with or without cigarette smoking. Pulmonary artery smooth muscle cells were cultured to study signaling pathways. Pulmonary hypertension phenotypes were evaluated in wild-type and calcium-sensing receptor knockout rats to identify the pathophysiological significance of the microRNA pathway. RESULTS: MicroR-1249 was predominant highly expressed in eEVs from plasma of rats exposed to cigarette smoking, and confirmed in eEVs from plasma of human smokers as well as in eEVs from cigarette smoke extract-treated pulmonary artery endothelial cells, but not in cigarette smoke extract-treated pulmonary artery smooth muscle cells. In cultured pulmonary artery smooth muscle cells, microR-1249 downregulated the expression of histone deacetylase 10, which in turn enhanced the acetylated form of NFκB (nuclear factor κB) level and its nuclear translocation leading to increased expression of calcium-sensing receptor. In rats, the repression of microR-1249 in eEVs by microR-1249 inhibitor, histone deacetylase 10 overexpression, or calcium-sensing receptor knockout profoundly inhibited the proliferative capacities and diminished apoptosis-resistance of pulmonary artery smooth muscle cells and pulmonary hypertension development in rats intravenously administrated with eEVs preparation from cigarette smoke extract-treated pulmonary artery endothelial cells. CONCLUSIONS: Cigarette smoke-enriched microR-1249 in endothelial extracellular vesicles facilitates the hyperproliferative and antiapoptotic status of pulmonary artery smooth muscle cells promoting pulmonary hypertension evolution through the inhibition of histone deacetylase 10-NFκB-calcium-sensing receptor cascade.

Guidelines on models of diabetic heart disease.

Diabetes is a major risk factor for cardiovascular diseases, including diabetic cardiomyopathy, atherosclerosis, myocardial infarction, and heart failure. As cardiovascular disease represents the number one cause of death in people with diabetes, there has been a major emphasis on understanding the mechanisms by which diabetes promotes cardiovascular disease, and how antidiabetic therapies impact diabetic heart disease. With a wide array of models to study diabetes (both type 1 and type 2), the field has made major progress in answering these questions. However, each model has its own inherent limitations. Therefore, the purpose of this guidelines document is to provide the field with information on which aspects of cardiovascular disease in the human diabetic population are most accurately reproduced by the available models. This review aims to emphasize the advantages and disadvantages of each model, and to highlight the practical challenges and technical considerations involved. We will review the preclinical animal models of diabetes (based on their method of induction), appraise models of diabetes-related atherosclerosis and heart failure, and discuss in vitro models of diabetic heart disease. These guidelines will allow researchers to select the appropriate model of diabetic heart disease, depending on the specific research question being addressed.

Calcium Sensing Receptor Variants Increase Pulmonary Hypertension Susceptibility.

BACKGROUND: Pulmonary arterial hypertension is an incurable disease, in which the extracellular CaSR (calcium sensing receptor) is mechanistically important. This study was aimed to genetically link the CaSR gene and function to the disease severity. METHODS: Sanger sequencing, Sugen/hypoxia pulmonary arterial hypertension rat model, CaSR mutated rat, transcriptional reporter assay and measurement of CaSR activity were used. RESULTS: Sanger sequencing identified a significant association between the variant rs1042636(A>G), located in CaSR exon 7, and idiopathic pulmonary arterial hypertension (IPAH) formation in patients. The frequency of 2968G homozygotes was higher in patients with IPAH compared with healthy individuals (23.6% versus 17.5%; P=0.001, OR=1.864), and the minor alleles of rs6776158, rs1048213, and rs9883099, located in CaSR promoter, raised the IPAH odds ratio to 2.173. Patients with IPAH carrying heterozygotes or homozygotes genotype of rs1042636 showed markedly higher pulmonary artery pressure and reduced survival compared with individuals carrying the wild-type allele. The minor alleles of rs6776158, rs1048213, and rs9883099 increased CaSR expression in reporter assay. In Sugen/hypoxia pulmonary arterial hypertension rats, the point mutation replicating rs1042636 found in IPAH exacerbated pulmonary arterial hypertension severity by promoting the overexpression and the enhanced activity of CaSR. CONCLUSIONS: Our functional genomic analysis thus indicates that the CaSR minor alleles of rs1042636, rs6776158, rs1048213, and rs9883099 contribute to the development and severity of IPAH. These findings may benefit clinical prognosis and treatment for IPAH.

Very-Long-Chain Unsaturated Sphingolipids Mediate Oleate-Induced Rat ß-Cell Proliferation.

Fatty acid (FA) signaling contributes to ß-cell mass expansion in response to nutrient excess, but the underlying mechanisms are poorly understood. In the presence of elevated glucose, FA metabolism is shifted toward synthesis of complex lipids, including sphingolipids. Here, we tested the hypothesis that sphingolipids are involved in the ß-cell proliferative response to FA. Isolated rat islets were exposed to FA and 16.7 mmol/L glucose for 48-72 h, and the contribution of the de novo sphingolipid synthesis pathway was tested using the serine palmitoyltransferase inhibitor myriocin, the sphingosine kinase (SphK) inhibitor SKI II, or knockdown of SphK, fatty acid elongase 1 (ELOVL1) and acyl-CoA-binding protein (ACBP). Rats were infused with glucose and the lipid emulsion ClinOleic and received SKI II by gavage. ß-Cell proliferation was assessed by immunochemistry or flow cytometry. Sphingolipids were analyzed by liquid chromatography-tandem mass spectrometry. Among the FAs tested, only oleate increased ß-cell proliferation. Myriocin, SKI II, and SphK knockdown all decreased oleate-induced ß-cell proliferation. Oleate exposure did not increase the total amount of sphingolipids but led to a specific rise in 24:1 species. Knockdown of ACBP or ELOVL1 inhibited oleate-induced ß-cell proliferation. We conclude that unsaturated very-long-chain sphingolipids produced from the available C24:1 acyl-CoA pool mediate oleate-induced ß-cell proliferation in rats.

Downregulation of low-density lipoprotein receptor mRNA in lymphatic endothelial cells impairs lymphatic function through changes in intracellular lipids.

Rationale: Impairment in lymphatic transport is associated with the onset and progression of atherosclerosis in animal models. The downregulation of low-density-lipoprotein receptor (LDLR) expression, rather than increased circulating cholesterol level per se, is involved in early atherosclerosis-related lymphatic dysfunction. Enhancing lymphatic function in Ldlr-/- mice with a mutant form of VEGF-C (VEGF-C 152s), a selective VEGFR-3 agonist, successfully delayed atherosclerotic plaque onset when mice were subsequently fed a high-fat diet. However, the specific mechanisms by which LDLR protects against lymphatic function impairment is unknown. Methods and results: We have thus injected wild-type and Pcsk9-/- mice with an adeno-associated virus type 1 expressing a shRNA for silencing Ldlr in vivo. We herein report that lymphatic contractility is reduced upon Ldlr dowregulation in wild-type mice only. Our in vitro experiments reveal that a decrease in LDLR expression at the mRNA level reduces the chromosome duplication phase and the protein expression of VEGFR-3, a membrane-bound key lymphatic marker. Furthermore, it also significantly reduced the levels of 18 lipid subclasses, including key constituents of lipid rafts as well as the transcription of several genes involved in cholesterol biosynthesis and cellular and metabolic processes. Exogenous PCSK9 only reduces lymphatic endothelial-LDLR at the protein level and does not affect lymphatic endothelial cell integrity. This puts forward that PCSK9 may act upon lymphatic muscle cells to mediate its effect on lymphatic contraction capacity in vivo. Conclusion: Our results suggest that treatments that specifically palliate the down regulation of LDLR mRNA in lymphatic endothelial cells preserve the integrity of the lymphatic endothelium and sustain lymphatic function, a prerequisite player in atherosclerosis.

Peptide Blocking Self-Polymerization of Extracellular Calcium-Sensing Receptor Attenuates Hypoxia-Induced Pulmonary Hypertension.

[Figure: see text].

Adaptive optimization of the OXPHOS assembly line partially compensates lrpprc-dependent mitochondrial translation defects in mice.

Mouse models of genetic mitochondrial disorders are generally used to understand specific molecular defects and their biochemical consequences, but rarely to map compensatory changes allowing survival. Here we took advantage of the extraordinary mitochondrial resilience of hepatic Lrpprc knockout mice to explore this question using native proteomics profiling and lipidomics. In these mice, low levels of the mtRNA binding protein LRPPRC induce a global mitochondrial translation defect and a severe reduction (>80%) in the assembly and activity of the electron transport chain (ETC) complex IV (CIV). Yet, animals show no signs of overt liver failure and capacity of the ETC is preserved. Beyond stimulation of mitochondrial biogenesis, results show that the abundance of mitoribosomes per unit of mitochondria is increased and proteostatic mechanisms are induced in presence of low LRPPRC levels to preserve a balance in the availability of mitochondrial- vs nuclear-encoded ETC subunits. At the level of individual organelles, a stabilization of residual CIV in supercomplexes (SCs) is observed, pointing to a role of these supramolecular arrangements in preserving ETC function. While the SC assembly factor COX7A2L could not contribute to the stabilization of CIV, important changes in membrane glycerophospholipid (GPL), most notably an increase in SC-stabilizing cardiolipins species (CLs), were observed along with an increased abundance of other supramolecular assemblies known to be stabilized by, and/or participate in CL metabolism. Together these data reveal a complex in vivo network of molecular adjustments involved in preserving mitochondrial integrity in energy consuming organs facing OXPHOS defects, which could be therapeutically exploited.

Differential modulation of polyunsaturated fatty acids in patients with myocardial infarction treated with ticagrelor or clopidogrel.

Untargeted metabolomics is used to refine the development of biomarkers for the diagnosis of cardiovascular disease. Myocardial infarction (MI) has major individual and societal consequences for patients, who remain at high risk of secondary events, despite advances in pharmacological therapy. To monitor their differential response to treatment, we performed untargeted plasma metabolomics on 175 patients from the platelet inhibition and patient outcomes (PLATO) trial treated with ticagrelor and clopidogrel, two common P2Y12 inhibitors. We identified a signature that discriminates patients, which involves polyunsaturated fatty acids (PUFAs) and particularly the omega-3 fatty acids docosahexaenoate and eicosapentaenoate. The known cardiovascular benefits of PUFAs could contribute to the efficacy of ticagrelor. Our work, beyond pointing out the high relevance of untargeted metabolomics in evaluating response to treatment, establishes PUFA metabolism as a pathway of clinical interest in the recovery path from MI.

Long-Chain Acylcarnitines and Monounsaturated Fatty Acids Discriminate Heart Failure Patients According to Pulmonary Hypertension Status.

Defects in fatty acid (FA) utilization have been well described in group 1 pulmonary hypertension (PH) and in heart failure (HF), yet poorly studied in group 2 PH. This study was to assess whether the metabolomic profile of patients with pulmonary hypertension (PH) due HF, classified as group 2 PH, differs from those without PH. We conducted a proof-of-principle cross-sectional analysis of 60 patients with chronic HF with reduced ejection fraction and 72 healthy controls in which the circulating level of 71 energy-related metabolites was measured using various methods. Echocardiography was used to classify HF patients as noPH-HF (n = 27; mean pulmonary artery pressure [mPAP] 21 mmHg) and PH-HF (n = 33; mPAP 35 mmHg). The profile of circulating metabolites among groups was compared using principal component analysis (PCA), analysis of covariance (ANCOVA), and Pearson's correlation tests. Patients with noPH-HF and PH-HF were aged 64 ± 11 and 68 ± 10 years, respectively, with baseline left ventricular ejection fractions of 27 ± 7% and 26 ± 7%. Principal component analysis segregated groups, more markedly for PH-HF, with long-chain acylcarnitines, acetylcarnitine, and monounsaturated FA carrying the highest loading scores. After adjustment for age, sex, kidney function, insulin resistance, and N-terminal pro-brain natriuretic peptide (NT-proBNP), 5/15 and 8/15 lipid-related metabolite levels were significantly different from controls in noPH-HF and PH-HF subjects, respectively. All metabolites for which circulating levels interacted between group and NT-proBNP significantly correlated with NT-proBNP in HF-PH, but none with HF-noPH. FA-related metabolites were differently affected in HF with or without PH, and may convey adverse outcomes given their distinct correlation with NT-proBNP in the setting of PH.

Dietary Geranylgeranyl Pyrophosphate Counteracts the Benefits of Statin Therapy in Experimental Pulmonary Hypertension.

BACKGROUND: The mevalonate pathway generates endogenous cholesterol and intermediates including geranylgeranyl pyrophosphate (GGPP). By reducing GGPP production, statins exert pleiotropic or cholesterol-independent effects. The potential regulation of GGPP homeostasis through dietary intake and the interaction with concomitant statin therapy is unknown. METHODS: We developed a sensitive high-pressure liquid chromatography technique to quantify dietary GGPP and conducted proteomics, qualitative real-time polymerase chain reaction screening, and Western blot to determine signaling cascades, gene expression, protein-protein interaction, and protein membrane trafficking in wild-type and transgenic rats. RESULTS: GGPP contents were highly variable depending on food source that differentially regulated blood GGPP levels in rats. Diets containing intermediate and high GGPP reduced or abolished the effects of statins in rats with hypoxia- and monocrotaline-induced pulmonary hypertension: this was rescuable by methyl-allylthiosulfinate and methyl-allylthiosulfinate-rich garlic extracts. In human pulmonary artery smooth muscle cells treated with statins, hypoxia activated RhoA in an extracellular GGPP-dependent manner. Hypoxia-induced ROCK2 (Rho associated coiled-coil containing protein kinase 2)/Rab10 (Ras-related protein rab-10) signaling was prevented by statin and recovered by exogenous GGPP. The hypoxia-activated RhoA/ROCK2 pathway in rat and human pulmonary artery smooth muscle cells upregulated the expression of Ca2+-sensing receptor (CaSR) and HIMF (hypoxia-induced mitogenic factor), a mechanism attenuated by statin treatment and regained with exogenous GGPP. Rab10 knockdown almost abrogated hypoxia-promoted CaSR membrane trafficking, a process diminished by statin and resumed by exogenous GGPP. Hypoxia-induced pulmonary hypertension was reduced in rats with CaSR mutated at the binding motif of HIMF and the interaction between dietary GGPP and statin efficiency was abolished. In humans fed a high GGPP diet, blood GGPP levels were increased. This abolished statin-lowering effects on plasma GGPP, and also on hypoxia-enhanced RhoA activity of blood monocytes that was rescued by garlic extracts. CONCLUSIONS: There is important dietary regulation of GGPP levels that interferes with the effects of statin therapy in experimental pulmonary hypertension. These observations rely on a key and central role of RhoA-ROCK2 cascade activation and Rab10-faciliated CaSR membrane trafficking with subsequent overexpression and binding of HIMF to CaSR. These findings warrant clinical investigation for the treatment of pulmonary hypertension and perhaps other diseases by combining statin with garlic-derived methyl-allylthiosulfinate or garlic extracts and thus circumventing dietary GGPP variations.