Neutrophil trafficking to the site of infection requires Cpt1a-dependent fatty acid ß-oxidation.

Cellular metabolism influences immune cell function, with mitochondrial fatty acid ß-oxidation and oxidative phosphorylation required for multiple immune cell phenotypes. Carnitine palmitoyltransferase 1a (Cpt1a) is considered the rate-limiting enzyme for mitochondrial metabolism of long-chain fatty acids, and Cpt1a deficiency is associated with infant mortality and infection risk. This study was undertaken to test the hypothesis that impairment in Cpt1a-dependent fatty acid oxidation results in increased susceptibility to infection. Screening the Cpt1a gene for common variants predicted to affect protein function revealed allele rs2229738_T, which was associated with pneumonia risk in a targeted human phenome association study. Pharmacologic inhibition of Cpt1a increases mortality and impairs control of the infection in a murine model of bacterial pneumonia. Susceptibility to pneumonia is associated with blunted neutrophilic responses in mice and humans that result from impaired neutrophil trafficking to the site of infection. Chemotaxis responsible for neutrophil trafficking requires Cpt1a-dependent mitochondrial fatty acid oxidation for amplification of chemoattractant signals. These findings identify Cpt1a as a potential host determinant of infection susceptibility and demonstrate a requirement for mitochondrial fatty acid oxidation in neutrophil biology.

Using genetics to detangle the relationships between red cell distribution width and cardiovascular diseases: a unique role for body mass index.

OBJECTIVE: Red cell distribution width (RDW) is an enigmatic biomarker associated with the presence and severity of multiple cardiovascular diseases (CVDs). It is unclear whether elevated RDW contributes to, results from, or is pleiotropically related to CVDs. We used contemporary genetic techniques to probe for evidence of aetiological associations between RDW, CVDs, and CVD risk factors. METHODS: Using an electronic health record (EHR)-based cohort, we built and deployed a genetic risk score (GRS) for RDW to test for shared genetic architecture between RDW and the cardiovascular phenome. We also created GRSs for common CVDs (coronary artery disease, heart failure, atrial fibrillation, peripheral arterial disease, venous thromboembolism) and CVD risk factors (body mass index (BMI), low-density lipoprotein, high-density lipoprotein, systolic blood pressure, diastolic blood pressure, serum triglycerides, estimated glomerular filtration rate, diabetes mellitus) to test each for association with RDW. Significant GRS associations were further interrogated by two-sample Mendelian randomisation (MR). In a separate EHR-based cohort, RDW values from 1-year pre-gastric bypass surgery and 1-2 years post-gastric bypass surgery were compared. RESULTS: In a cohort of 17 937 subjects, there were no significant associations between the RDW GRS and CVDs. Of the CVDs and CVD risk factors, only genetically predicted BMI was associated with RDW. In subsequent analyses, BMI was associated with RDW by multiple MR methods. In subjects undergoing bariatric surgery, RDW decreased postsurgery and followed a linear relationship with BMI change. CONCLUSIONS: RDW is unlikely to be aetiologically upstream or downstream of CVDs or CVD risk factors except for BMI. Genetic and clinical association analyses support an aetiological relationship between BMI and RDW.

BMI Is Causally Associated With Pulmonary Artery Pressure But Not Hemodynamic Evidence of Pulmonary Vascular Remodeling.

BACKGROUND: There is an unclear relationship of obesity to the pathogenesis and severity of pulmonary arterial hypertension (PAH) and pulmonary venous hypertension (PVH). RESEARCH QUESTION: Is BMI casually associated with pulmonary artery pressure (PAP) and/or markers of pulmonary vascular remodeling? STUDY DESIGN AND METHODS: The study design was a two-sample inverse-variance weighted Mendelian randomization. We constructed two BMI genetic risk scores from genome-wide association study summary data and deployed them in nonoverlapping cohorts of subjects referred for right heart catheterization (RHC) or echocardiography. A BMI highly polygenic risk score (hpGRS) optimally powered to detect shared genetic architecture of obesity with other traits was tested for association with RHC parameters including markers of pulmonary vascular remodeling. A BMI strict genetic risk score (sGRS) composed of high-confidence genetic variants was used for Mendelian randomization analyses to assess if higher BMI causes higher PAP. RESULTS: Among all subjects, both directly measured BMI and hpGRS were positively associated with pulmonary arterial pressures but not markers of pulmonary vascular remodeling. Categorical analyses revealed BMI and hpGRS were associated with PVH but not PAH. Mendelian randomization of the sGRS supported that higher BMI is causal of higher systolic pulmonary artery pressure (sPAP). Sensitivity analyses showed sPAP-BMI sGRS relationship was preserved when either individuals with PAH or PVH were excluded. In the echocardiographic cohort, BMI and hpGRS were positively associated with estimated PAP and markers of left heart remodeling. INTERPRETATION: BMI is a modifier of pulmonary hypertension severity in both PAH and PVH but is only involved in the pathogenesis of PVH.

The Role of Bone Morphogenetic Protein Signaling in Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) affects over 30% of adults in the United States. Bone morphogenetic protein (BMP) signaling is known to contribute to hepatic fibrosis, but the role of BMP signaling in the development of NAFLD is unclear. In this study, treatment with either of two BMP inhibitors reduced hepatic triglyceride content in diabetic (db/db) mice. BMP inhibitor-induced decrease in hepatic triglyceride levels was associated with decreased mRNA encoding Dgat2, an enzyme integral to triglyceride synthesis. Treatment of hepatoma cells with BMP2 induced DGAT2 expression and activity via intracellular SMAD signaling. In humans we identified a rare missense single nucleotide polymorphism in the BMP type 1 receptor ALK6 (rs34970181;R371Q) associated with a 2.1-fold increase in the prevalence of NAFLD. In vitro analyses revealed R371Q:ALK6 is a previously unknown constitutively active receptor. These data show that BMP signaling is an important determinant of NAFLD in a murine model and is associated with NAFLD in humans.

Mendelian randomisation analysis of red cell distribution width in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a rare disease that leads to premature death from right heart failure. It is strongly associated with elevated red cell distribution width (RDW), a correlate of several iron status biomarkers. High RDW values can signal early-stage iron deficiency or iron deficiency anaemia. This study investigated whether elevated RDW is causally associated with PAH.A two-sample Mendelian randomisation (MR) approach was applied to investigate whether genetic predisposition to higher levels of RDW increases the odds of developing PAH. Primary and secondary MR analyses were performed using all available genome-wide significant RDW variants (n=179) and five genome-wide significant RDW variants that act via systemic iron status, respectively.We confirmed the observed association between RDW and PAH (OR 1.90, 95% CI 1.80-2.01) in a multicentre case-control study (cases n=642, disease controls n=15 889). The primary MR analysis was adequately powered to detect a causal effect (odds ratio) between 1.25 and 1.52 or greater based on estimates reported in the RDW genome-wide association study or from our own data. There was no evidence for a causal association between RDW and PAH in either the primary (ORcausal 1.07, 95% CI 0.92-1.24) or the secondary (ORcausal 1.09, 95% CI 0.77-1.54) MR analysis.The results suggest that at least some of the observed association of RDW with PAH is secondary to disease progression. Results of iron therapeutic trials in PAH should be interpreted with caution, as any improvements observed may not be mechanistically linked to the development of PAH.

Unbiased Phenome-Wide Association Studies of Red Cell Distribution Width Identifies Key Associations with Pulmonary Hypertension.

Rationale: Red cell distribution width (RDW) is a prognostic factor in many diseases; however, its clinical utility remains limited because the relative value of RDW as a biomarker across disease states has not been established. Objectives: To establish an unbiased RDW disease hierarchy to guide the clinical use of RDW and to assess its relationship to cardiovascular hemodynamic and structural parameters. Methods: We performed phenome-wide association studies for RDW in discovery and replication cohorts derived from a deidentified electronic health record in nonanemic individuals. RDW values obtained within 30 days of echocardiogram or right heart catheterization were tested for association with structural and hemodynamic variables. Results: RDW was associated with 263 phenotypes in both men and women in the discovery cohort (n = 121,530), 48 of which replicated in an independent cohort (n = 2,039). The strongest associations were observed with pulmonary arterial hypertension (odds ratio [OR], 2.1; 95% confidence interval [CI], 1.9-2.3), chronic pulmonary heart disease (OR, 2.0; 95% CI, 1.9-2.2), and congestive heart failure (OR, 1.9; 95% CI, 1.8-2.0); P < 1 × 10-74 for all. By echocardiography, RDW was higher in the setting of right ventricular dysfunction than left ventricular dysfunction (P < 0.001). Measured invasively, mean pulmonary arterial pressure was associated with RDW (21 vs. 33 mm Hg at 25th vs. 75th percentile RDW; P < 1 × 10-7) and remained strongly significant even when controlling for mean pulmonary capillary wedge pressure (21 vs. 29 mm Hg at 25th vs. 75th percentile RDW; P < 1 × 10-7). Conclusions: Among 1,364 coded medical conditions, increased RDW was strongly associated with pulmonary hypertension and heart failure. Hemodynamic and echocardiographic phenotyping confirmed these associations and underscored that the most clinically relevant phenotype associated with RDW was pulmonary hypertension. These hypothesis-generating findings highlight the potential shared pathophysiology of pulmonary hypertension and elevated RDW. Elevated RDW in the absence of anemia should alert clinicians to the potential for underlying cardiopulmonary disease.

Clinical and genetic associations with prostacyclin response in pulmonary arterial hypertension.

Parenteral prostacyclin therapy is the most efficacious pharmacologic treatment for pulmonary arterial hypertension (PAH), but clinical response is variable. We sought to identify clinical, hemodynamic, and genetic associations with response to prostacyclin therapy. We performed a retrospective analysis of patients within a de-identified electronic health record and associated DNA biobank. Patients with PAH and a right heart catheterization (RHC) in the six months before initiation of a parenteral prostacyclin were included. Responders were defined a priori by attainment of World Health Organization (WHO) functional class (FC) 2 or better at the time of repeat RHC within two years. We performed exploratory analyses to identify genomic associations with prostacyclin response. Of 129 patients identified, 54 met our criteria for "responders." These patients were younger, more likely to be male, and were less likely to have connective tissue disease-related PAH. At follow-up, responders had improved hemodynamics, 6-min walk distance, and long-term survival. Baseline PA oxygen saturation (hazard ratio [HR] 0.568 [0.34-0.95]) and follow-up FC (HR = 2.57 [1.22-5.43]) were associated with survival. Prostacyclin responders were enriched in alleles related to cell development and circulatory system development and pathways related to aldosterone metabolism, cAMP signaling, and vascular smooth muscle contraction ( P < 0.001). Age at treatment initiation, WHO FC at short-term follow-up, and PA O2% are associated with survival in patients with PAH exposed to parenteral prostacyclins. Exploratory genetic analysis yielded associations in biologically relevant pathways in the pathogenesis of PAH.

Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation.

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Atherosclerotic plaques, consisting of lipid-laden macrophages and calcification, develop in the coronary arteries, aortic valve, aorta, and peripheral conduit arteries and are the hallmark of cardiovascular disease. In humans, imaging with computed tomography allows for the quantification of vascular calcification; the presence of vascular calcification is a strong predictor of future cardiovascular events. Development of novel therapies in cardiovascular disease relies critically on improving our understanding of the underlying molecular mechanisms of atherosclerosis. Advancing our knowledge of atherosclerotic mechanisms relies on murine and cell-based models. Here, a method for imaging aortic calcification and macrophage infiltration using two spectrally distinct near-infrared fluorescent imaging probes is detailed. Near-infrared fluorescent imaging allows for the ex vivo quantification of calcification and macrophage accumulation in the entire aorta and can be used to further our understanding of the mechanistic relationship between inflammation and calcification in atherosclerosis. Additionally, a method for isolating and culturing animal aortic vascular smooth muscle cells and a protocol for inducing calcification in cultured smooth muscle cells from either murine aortas or from human coronary arteries is described. This in vitro method of modeling vascular calcification can be used to identify and characterize the signaling pathways likely important for the development of vascular disease, in the hopes of discovering novel targets for therapy.

Inhibition of bone morphogenetic protein signal transduction prevents the medial vascular calcification associated with matrix Gla protein deficiency.

OBJECTIVE: Matrix Gla protein (MGP) is reported to inhibit bone morphogenetic protein (BMP) signal transduction. MGP deficiency is associated with medial calcification of the arterial wall, in a process that involves both osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs) and mesenchymal transition of endothelial cells (EndMT). In this study, we investigated the contribution of BMP signal transduction to the medial calcification that develops in MGP-deficient mice. APPROACH AND RESULTS: MGP-deficient mice (MGP(-/-)) were treated with one of two BMP signaling inhibitors, LDN-193189 or ALK3-Fc, beginning one day after birth. Aortic calcification was assessed in 28-day-old mice by measuring the uptake of a fluorescent bisphosphonate probe and by staining tissue sections with Alizarin red. Aortic calcification was 80% less in MGP(-/-) mice treated with LDN-193189 or ALK3-Fc compared with vehicle-treated control animals (P<0.001 for both). LDN-193189-treated MGP(-/-) mice survived longer than vehicle-treated MGP(-/-) mice. Levels of phosphorylated Smad1/5 and Id1 mRNA (markers of BMP signaling) did not differ in the aortas from MGP(-/-) and wild-type mice. Markers of EndMT and osteogenesis were increased in MGP(-/-) aortas, an effect that was prevented by LDN-193189. Calcification of isolated VSMCs was also inhibited by LDN-193189. CONCLUSIONS: Inhibition of BMP signaling leads to reduced vascular calcification and improved survival in MGP(-/-) mice. The EndMT and osteogenic transdifferentiation associated with MGP deficiency is dependent upon BMP signaling. These results suggest that BMP signal transduction has critical roles in the development of vascular calcification in MGP-deficient mice.