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The complex between lipoprotein lipase (LPL) and its endothelial receptor (GPIHBP1) is responsible for the lipolytic processing of triglyceride-rich lipoproteins (TRLs) along the capillary lumen, a physiologic process that releases lipid nutrients for vital organs such as heart and skeletal muscle. LPL activity is regulated in a tissue-specific manner by endogenous inhibitors (angiopoietin-like [ANGPTL] proteins 3, 4, and 8), but the molecular mechanisms are incompletely understood. ANGPTL4 catalyzes the inactivation of LPL monomers by triggering the irreversible unfolding of LPL's α/β-hydrolase domain. Here, we show that this unfolding is initiated by the binding of ANGPTL4 to sequences near LPL's catalytic site, including β2, β3-α3, and the lid. Using pulse-labeling hydrogen‒deuterium exchange mass spectrometry, we found that ANGPTL4 binding initiates conformational changes that are nucleated on β3-α3 and progress to β5 and β4-α4, ultimately leading to the irreversible unfolding of regions that form LPL's catalytic pocket. LPL unfolding is context dependent and varies with the thermal stability of LPL's α/β-hydrolase domain (Tm of 34.8 °C). GPIHBP1 binding dramatically increases LPL stability (Tm of 57.6 °C), while ANGPTL4 lowers the onset of LPL unfolding by ∼20 °C, both for LPL and LPL•GPIHBP1 complexes. These observations explain why the binding of GPIHBP1 to LPL retards the kinetics of ANGPTL4-mediated LPL inactivation at 37 °C but does not fully suppress inactivation. The allosteric mechanism by which ANGPTL4 catalyzes the irreversible unfolding and inactivation of LPL is an unprecedented pathway for regulating intravascular lipid metabolism.

The binding of lipoprotein lipase (LPL) to GPIHBP1 focuses the intravascular hydrolysis of triglyceride-rich lipoproteins on the surface of capillary endothelial cells. This process provides essential lipid nutrients for vital tissues (e.g., heart, skeletal muscle, and adipose tissue). Deficiencies in either LPL or GPIHBP1 impair triglyceride hydrolysis, resulting in severe hypertriglyceridemia. The activity of LPL in tissues is regulated by angiopoietin-like proteins 3, 4, and 8 (ANGPTL). Dogma has held that these ANGPTLs inactivate LPL by converting LPL homodimers into monomers, rendering them highly susceptible to spontaneous unfolding and loss of enzymatic activity. Here, we show that binding of an LPL-specific monoclonal antibody (5D2) to the tryptophan-rich lipid-binding loop in the carboxyl terminus of LPL prevents homodimer formation and forces LPL into a monomeric state. Of note, 5D2-bound LPL monomers are as stable as LPL homodimers (i.e., they are not more prone to unfolding), but they remain highly susceptible to ANGPTL4-catalyzed unfolding and inactivation. Binding of GPIHBP1 to LPL alone or to 5D2-bound LPL counteracts ANGPTL4-mediated unfolding of LPL. In conclusion, ANGPTL4-mediated inactivation of LPL, accomplished by catalyzing the unfolding of LPL, does not require the conversion of LPL homodimers into monomers. Thus, our findings necessitate changes to long-standing dogma on mechanisms for LPL inactivation by ANGPTL proteins. At the same time, our findings align well with insights into LPL function from the recent crystal structure of the LPL•GPIHBP1 complex.

The intravascular processing of triglyceride-rich lipoproteins depends on lipoprotein lipase (LPL) and GPIHBP1, a membrane protein of endothelial cells that binds LPL within the subendothelial spaces and shuttles it to the capillary lumen. In the absence of GPIHBP1, LPL remains mislocalized within the subendothelial spaces, causing severe hypertriglyceridemia (chylomicronemia). The N-terminal domain of GPIHBP1, an intrinsically disordered region (IDR) rich in acidic residues, is important for stabilizing LPL's catalytic domain against spontaneous and ANGPTL4-catalyzed unfolding. Here, we define several important properties of GPIHBP1's IDR. First, a conserved tyrosine in the middle of the IDR is posttranslationally modified by O-sulfation; this modification increases both the affinity of GPIHBP1-LPL interactions and the ability of GPIHBP1 to protect LPL against ANGPTL4-catalyzed unfolding. Second, the acidic IDR of GPIHBP1 increases the probability of a GPIHBP1-LPL encounter via electrostatic steering, increasing the association rate constant (kon) for LPL binding by >250-fold. Third, we show that LPL accumulates near capillary endothelial cells even in the absence of GPIHBP1. In wild-type mice, we expect that the accumulation of LPL in close proximity to capillaries would increase interactions with GPIHBP1. Fourth, we found that GPIHBP1's IDR is not a key factor in the pathogenicity of chylomicronemia in patients with the GPIHBP1 autoimmune syndrome. Finally, based on biophysical studies, we propose that the negatively charged IDR of GPIHBP1 traverses a vast space, facilitating capture of LPL by capillary endothelial cells and simultaneously contributing to GPIHBP1's ability to preserve LPL structure and activity.

Lipoprotein lipase (LPL) undergoes spontaneous inactivation via global unfolding and this unfolding is prevented by GPIHBP1 (Mysling et al., 2016). We now show: (1) that ANGPTL4 inactivates LPL by catalyzing the unfolding of its hydrolase domain; (2) that binding to GPIHBP1 renders LPL largely refractory to this inhibition; and (3) that both the LU domain and the intrinsically disordered acidic domain of GPIHBP1 are required for this protective effect. Genetic studies have found that a common polymorphic variant in ANGPTL4 results in lower plasma triglyceride levels. We now report: (1) that this ANGPTL4 variant is less efficient in catalyzing the unfolding of LPL; and (2) that its Glu-to-Lys substitution destabilizes its N-terminal α-helix. Our work elucidates the molecular basis for regulation of LPL activity by ANGPTL4, highlights the physiological relevance of the inherent instability of LPL, and sheds light on the molecular defects in a clinically relevant variant of ANGPTL4.

GPIHBP1 is a glycolipid-anchored membrane protein of capillary endothelial cells that binds lipoprotein lipase (LPL) within the interstitial space and shuttles it to the capillary lumen. The LPL•GPIHBP1 complex is responsible for margination of triglyceride-rich lipoproteins along capillaries and their lipolytic processing. The current work conceptualizes a model for the GPIHBP1•LPL interaction based on biophysical measurements with hydrogen-deuterium exchange/mass spectrometry, surface plasmon resonance, and zero-length cross-linking. According to this model, GPIHBP1 comprises two functionally distinct domains: (1) an intrinsically disordered acidic N-terminal domain; and (2) a folded C-terminal domain that tethers GPIHBP1 to the cell membrane by glycosylphosphatidylinositol. We demonstrate that these domains serve different roles in regulating the kinetics of LPL binding. Importantly, the acidic domain stabilizes LPL catalytic activity by mitigating the global unfolding of LPL's catalytic domain. This study provides a conceptual framework for understanding intravascular lipolysis and GPIHBP1 and LPL mutations causing familial chylomicronemia.

ICMA, a software framework to create 3D finite element models of the left ventricle from cardiac ultrasound or magnetic resonance imaging (MRI) data, has been made available as an open-source code. The framework is hardware vendor independent and uses spec

Lipoprotein lipase (LPL) undergoes spontaneous inactivation via global unfolding and this unfolding is prevented by GPIHBP1 (Mysling et al., 2016). We now show: (1) that ANGPTL4 inactivates LPL by catalyzing the unfolding of its hydrolase domain; (2) that binding to GPIHBP1 renders LPL largely refractory to this inhibition; and (3) that both the LU domain and the intrinsically disordered acidic domain of GPIHBP1 are required for this protective effect. Genetic studies have found that a common polymorphic variant in ANGPTL4 results in lower plasma triglyceride levels. We now report: (1) that this ANGPTL4 variant is less efficient in catalyzing the unfolding of LPL; and (2) that its Glu-to-Lys substitution destabilizes its N-terminal α-helix. Our work elucidates the molecular basis for regulation of LPL activity by ANGPTL4, highlights the physiological relevance of the inherent instability of LPL, and sheds light on the molecular defects in a clinically relevant variant of ANGPTL4.

GPIHBP1 is a glycolipid-anchored membrane protein of capillary endothelial cells that binds lipoprotein lipase (LPL) within the interstitial space and shuttles it to the capillary lumen. The LPL•GPIHBP1 complex is responsible for margination of triglyceride-rich lipoproteins along capillaries and their lipolytic processing. The current work conceptualizes a model for the GPIHBP1•LPL interaction based on biophysical measurements with hydrogen-deuterium exchange/mass spectrometry, surface plasmon resonance, and zero-length cross-linking. According to this model, GPIHBP1 comprises two functionally distinct domains: (1) an intrinsically disordered acidic N-terminal domain; and (2) a folded C-terminal domain that tethers GPIHBP1 to the cell membrane by glycosylphosphatidylinositol. We demonstrate that these domains serve different roles in regulating the kinetics of LPL binding. Importantly, the acidic domain stabilizes LPL catalytic activity by mitigating the global unfolding of LPL's catalytic domain. This study provides a conceptual framework for understanding intravascular lipolysis and GPIHBP1 and LPL mutations causing familial chylomicronemia.

Context: Previous studies using autologous bone marrow mononuclear cells (BMCs) in patients with ischemic cardiomyopathy have demonstrated safety and suggested efficacy.Objective: To determine if administration of BMCs through transendocardial injections improves myocardial perfusion, reduces left ventricular end-systolic volume (LVESV), or enhances maximal oxygen consumption in patients with coronary artery disease or LV dysfunction, and limiting heart failure or angina.Design, Setting, and Patients: A phase 2 randomized double-blind, placebo-controlled trial of symptomatic patients (New York Heart Association classification II-III or Canadian Cardiovascular Society classification II-IV) with a left ventricular ejection fraction of 45% or less, a perfusion defect by single-photon emission tomography (SPECT), and coronary artery disease not amenable to revascularization who were receiving maximal medical therapy at 5 National Heart, Lung, and Blood Institute-sponsored Cardiovascular Cell Therapy Research Network (CCTRN) sites between April 29, 2009, and April 18, 2011.Intervention: Bone marrow aspiration (isolation of BMCs using a standardized automated system performed locally) and transendocardial injection of 100 million BMCs or placebo (ratio of 2 for BMC group to 1 for placebo group).Main Outcome Measures: Co-primary end points assessed at 6 months: changes in LVESV assessed by echocardiography, maximal oxygen consumption, and reversibility on SPECT. Phenotypic and functional analyses of the cell product were performed by the CCTRN biorepository core laboratory.Results: Of 153 patients who provided consent, a total of 92 (82 men; average age: 63 years) were randomized (n = 61 in BMC group and n = 31 in placebo group). Changes in LVESV index (-0.9 mL/m(2) [95% CI, -6.1 to 4.3]; P = .73), maximal oxygen consumption (1.0 [95% CI, -0.42 to 2.34]; P = .17), and reversible defect (-1.2 [95% CI, -12.50 to 10.12]; P = .84) were not statistically significant. There were no differences found in any of the secondary outcomes, including percent myocardial defect, total defect size, fixed defect size, regional wall motion, and clinical improvement.Conclusion: Among patients with chronic ischemic heart failure, transendocardial injection of autologous BMCs compared with placebo did not improve LVESV, maximal oxygen consumption, or reversibility on SPECT.Trial Registration: clinicaltrials.gov Identifier: NCT00824005. [ABSTRACT FROM AUTHOR]

Objective: The present analysis evaluates the overall appreciation and implementation of an intervention, Project Tomato, designed to maintain fruit and vegetable intake in children aged 8-9 years.Design: A random sample of fifty-four English primary schools (658 children) were randomised to either the intervention group or the control. The intervention group received a multi-component programme delivered in school by teachers and items sent home for parents/children. Dietary measurements were collected at baseline and follow-up. The intervention participants completed questionnaires on the intervention materials, to identify implementation and appreciation of the intervention, and other environmental mechanisms.Setting: Fifty-four primary schools were randomly selected, with twenty-seven schools allocated to the intervention group.Subjects: A total of 311 children received the intervention.Results: Implementation of the intervention was low, 21·3 % of school items and 56·0 % of home items were implemented. The intervention materials were well received by teachers, parents and children. Other mechanisms that may affect fruit and vegetable intake were explored. Children who ate their main meal with their parents 3-7 nights/week on average consumed 37·6 (95 % CI 9·8, 65·4) g more fruit and vegetables than children who ate with their parents 0-2 times/week.Conclusions: Implementation of the trial components was poor. However, the results identified the importance of parental environment and mealtime structure on children's fruit and vegetable intake. [ABSTRACT FROM AUTHOR]

Background: Neutrophil gelatinase-associated lipocalin (NGAL) is released by renal tubular cells in response to inflammation and injury. Recent studies have demonstrated that NGAL is up-regulated in cardiomyocytes within the failing myocardium. However, the overall relationship between systemic NGAL levels and myocardial structure and performance has not been established.Methods and Results: We measured systemic NGAL levels in 130 subjects with chronic systolic heart failure (HF) and comprehensive echocardiographic evaluation, as well as 69 subjects with acute decompensated systolic HF and hemodynamic evaluation. In the chronic HF cohort, higher plasma NGAL levels were modestly associated with increasing age (r = 0.18; P = .035), higher New York Heart Association functional class (rank sums: P = .022) and impaired renal function (eGFR: r = -0.53; P < .0001; cystatin C: r = 0.60; P < .0001). Plasma NGAL levels were modestly associated with indices of diastolic dysfunction (mitral E/Ea: r = 0.27; P = .002; LAVi: r = 0.25; P = .011; tricuspid E/Ea: r = 0.20; P = .029), but not after adjustment for renal function (P > .10 for all). In Cox proportional hazards analysis, plasma NGAL predicted cardiac death or transplantation after adjustment for age, gender, left ventricular ejection fraction, and mitral E/Ea (hazard ratio 1.68, 95% confidence interval 1.08-2.57; P = .022), but not after adjustment for renal function (P = .83). In the acute HF cohort, we did not observe any relationship between NGAL and hemodynamic indices, but NGAL strongly correlated with renal function.Conclusions: Systemic NGAL levels are largely determined by underlying impairment of renal rather than myocardial function. Our findings did not support any prognostic significance or relationship between systemic NGAL levels and indices of cardiac structure and function after adjustment for underlying renal function. [ABSTRACT FROM AUTHOR]