Low-density lipoprotein aggregation predicts adverse cardiovascular events in peripheral artery disease

Background and aims: Peripheral artery disease (PAD) is a systemic manifestation of atherosclerosis that is associated with a high risk of major adverse cardiovascular events (MACE). LDL aggregation contributes to atherosclerotic plaque progression and may contribute to plaque instability. We aimed to determine if LDL aggregation is associated with MACE in patients with PAD undergoing lower extremity revascularization (LER). Methods: Two hundred thirty-nine patients with PAD undergoing LER had blood collected at baseline and were followed prospectively for MACE (myocardial infarction, stroke, cardiovascular death) for one year. Nineteen age, sex and LDL-C-matched control subjects without cardiovascular disease also had blood drawn. Subject LDL was exposed to sphingomyelinase and LDL aggregate size measured via dynamic light scattering. Results: Mean age was 72.3 10.9 years, 32.6% were female, and LDL-cholesterol was 68 +/- 25 mg/dL. LDL aggregation was inversely associated with triglycerides, but not associated with demographics, LDL-cholesterol or other risk factors. Maximal LDL aggregation occurred significantly earlier in subjects with PAD than in control subjects. 15.9% of subjects experienced MACE over one year. The 1st tertile (shortest time to maximal aggregation) exhibited significantly higher MACE (25% vs. 12.5% in tertile 2 and 10.1% in tertile 3, p = 0.012). After multivariable adjustment for demographics and CVD risk factors, the hazard ratio for MACE in the 1st tertile was 4.57 (95% CI 1.60-13.01; p = 0.004) compared to tertile 3. Inclusion of LDL aggregation in the Framingham Heart Study risk calculator for recurrent coronary heart disease events improved the c-index from 0.57 to 0.63 (p = 0.01). Conclusions: We show that in the setting of very well controlled LDL-cholesterol, patients with PAD with the most rapid LDL aggregation had a significantly elevated MACE risk following LER even after multivariable adjustment. This measure further improved the classification specificity of an established risk prediction tool. Our findings support broader investigation of this assay for risk stratification in patients with atherosclerotic CVD.Peer reviewe

Transmembrane Prostatic Acid Phosphatase (TMPAP) Interacts with Snapin and Deficient Mice Develop Prostate Adenocarcinoma

Peer reviewe

Overfeeding saturated fat increases LDL (low-density lipoprotein) aggregation susceptibility while overfeeding unsaturated fat decreases proteoglycan-binding of lipoproteins

Publisher Copyright: © 2021 The Authors.OBJECTIVE: We recently showed that measurement of the susceptibility of LDL (low-density lipoprotein) to aggregation is an independent predictor of cardiovascular events. We now wished to compare effects of overfeeding different dietary macronutrients on LDL aggregation, proteoglycan-binding of plasma lipoproteins, and on the concentration of oxidized LDL in plasma, 3 in vitro parameters consistent with increased atherogenicity. APPROACH AND RESULTS: The participants (36 subjects; age, 48±10 years; body mass index, 30.9±6.2 kg/m2) were randomized to consume an extra 1000 kcal/day of either unsaturated fat, saturated fat, or simple sugars (CARB) for 3 weeks. We measured plasma proatherogenic properties (susceptibility of LDL to aggregation, proteoglycan-binding, oxidized LDL) and concentrations and composition of plasma lipoproteins using nuclear magnetic resonance spectroscopy, and in LDL using liquid chromatography mass spectrometry, before and after the overfeeding diets. LDL aggregation increased in the saturated fat but not the other groups. This change was associated with increased sphingolipid and saturated triacylglycerols in LDL and in plasma and reduction of clusterin on LDL particles. Proteoglycan binding of plasma lipoproteins decreased in the unsaturated fat group relative to the baseline diet. Lipoprotein properties remained unchanged in the CARB group. CONCLUSIONS: The type of fat during 3 weeks of overfeeding is an important determinant of the characteristics and functional properties of plasma lipoproteins in humans.Peer reviewe

TMPAP co-localize and interact with snapin in the cell lamellipodia.

<p>A, co-localization (yellow) of TMPAP (green) with snapin (red) was observed in the vesicles and lamellipodia of the TMPAP/LNCaP cells. Arrows mark the co-localization points in the upper panel (scale bar: 20 µm). Lower panel (scale bar: 3 µm) showing the lamellipodia region, amplification of the area marked with a box in the upper panel (left). B, intensification of donor (TMPAP-GFP) fluorescence in LNCaP cells was observed after acceptor (snapin-DsRed) photobleaching which confirms FRET between two molecules (Scale bar: 2 µm).</p

DLP lobeexhibits the primary changes in the PAP^−/− mouse prostate.

<p>The panels show an overview of the 12-old mice prostate dissected lobes. The DLP, AP and VP lobes were dissected from WT and PAP^−/− mouse. The monolayer epithelium (white arrows) is seen in all the lobes of the WT mouse, whereas in the PAP^−/− mouse an increased amount of cells is present in the lumen of the DLP lobe (black arrows). The AP and VP of PAP^−/− mouse show no significant changes. Scale bars: 100 µm.</p

Significant ontological terms obtained with GoMiner software from two-color microarrays experiments.

<p><b>GO ID</b>: gene ontology ID accession number. <b><i>P</i></b><b>-value</b>: <i>P</i>-value for the number of changed genes in the input list, significant <i>P</i>-value <0.05. <b>Term</b>: associated ontological term. <b>Rows in bold</b>: relevant ontological groups for vesicular transport. Mice in microarray experiment per group, <i>n = </i>3.</p

Proliferation of DLP cells is increased in PAP^−/− mice compared to WT mice.

<p>A, bar plot showing the ratio (as percentage) between proliferative cell counts and total amount of cells. (**, P value <0.01; ***, P value <0.001). Error bars indicate S.E.M. values. B, bar plot showing the ratio (as percentage) between apoptotic cell counts and total amount of cells. Error bars indicate S.E.M. values.</p

The prostate adenocarcinoma in PAP^−/− mice is also detected by immunohistochemistry and electronmicroscopy.

<p>A, smooth muscle actin (SMA) immunohistochemistry in 12 month-old mice. Monolayer epithelium (mL) and open lumen in PAP^+/+ DLP. White arrows show the broken fibromuscular sheath (SM, smooth muscle) and bulging of epithelial cells to the stroma. Prostate adenocarcinoma (black arrows) is present in AP and DLP, showing a multilayer epithelium (ML) and inflammatory cells (black arrowhead) spreading in neighboring areas. Scale bars: 100 µm. (<i>n</i> =  4, per group). B, ultrastructural changes in 3 month-old and 12 month-old PAP^−/− mouse DLPs. Monolayer epithelium, regular basement membrane (BM) and apical secretion are clearly seen in PAP^+/+ mouse DLPs. 3 month-old PAP^−/− DLPs show irregular BM and numerous apical vacuoles (red arrow head), as well the presence of basal lysosomes (Ly). In 12 month-old PAP−/− mouse DLPs, the epithelium has transformed to a multilayer epithelium containing hyperchromatic nuclei with multiple nucleoli. Pseudolumens (pL) have formed as a result of the growing and fusion of the epithelium. Invaginations of BM (red arrows) into the epithelium and numerous vesicles in the basal side of the cells (blue arrow heads) were additional signs of the transformation in the cells. Scale bars: 2000 nm (<i>n</i> = 4, per group).</p

TMPAP is involved in endo-/exocytosis (proposed mechanism).

<p>TMPAP synthesized in the endoplasmic reticulum is transported in vesicles to the plasma membrane through the trans-Golgi network (TGN). After the vesicle docking and fusion events leading to release of vesicle content, TMPAP inserted in plasma membrane exerts its phosphatase function over AMP. The resulting product adenosine (Ado) activates the adenosine receptors, which are GPCRs, A1 or A3with G_αi (inhibitory G-protein β-subunit) specificity leading to the inhibition of adenylate cyclase (AC) activity, and A2 adenosine receptors with G_αs (stimulatory G-protein α-subunit) producing the stimulation of AC activity. Activated AC produces cAMP, which activates PKA responsible for the phosphorylation of snapin. The turnover is completed by clathrin-mediated endocytosis of SNARE components and TMPAP for recycling and degradation in lysosomes vía the endosomal-lysosomal pathway. From early endosomes, the cargo can be sorted to late endosomes or to MVE, which can follow the route leading to exosome release. Additional dephosphorylation events by TMPAP can occur while trafficking between different compartments. From late endosomes, TMPAP can go to lysosomes or back to TGN via the retrograde pathway. ATP: adenosine triphosphate, ADP: adenosine diphosphate, AMP: adenosine monophosphate, Ado: adenosine, TGN: trans-Golgi network, P: phosphate group, AP-2: adaptor protein complex 2, ADORA: adenosine receptor A (types A1, A2 and A3), AC: adenylate cyclase, G_αs, G_αi, G_β, G_γ: G-protein subunits, VDCC: Voltage-gated calcium channel. Synaptobrevin, syntaxin and SNAP25 are SNARE proteins. PI (4,5) P₂: phosphatidylinositol 4,5-bisphosphate.</p

Susceptibility of low-density lipoprotein particles to aggregate depends on particle lipidome, is modifiable, and associates with future cardiovascular deaths

Abstract Aims: Low-density lipoprotein (LDL) particles cause atherosclerotic cardiovascular disease (ASCVD) through their retention, modification, and accumulation within the arterial intima. High plasma concentrations of LDL drive this disease, but LDL quality may also contribute. Here, we focused on the intrinsic propensity of LDL to aggregate upon modification. We examined whether inter-individual differences in this quality are linked with LDL lipid composition and coronary artery disease (CAD) death, and basic mechanisms for plaque growth and destabilization. Methods and results: We developed a novel, reproducible method to assess the susceptibility of LDL particles to aggregate during lipolysis induced ex vivo by human recombinant secretory sphingomyelinase. Among patients with an established CAD, we found that the presence of aggregation-prone LDL was predictive of future cardiovascular deaths, independently of conventional risk factors. Aggregation-prone LDL contained more sphingolipids and less phosphatidylcholines than did aggregation-resistant LDL. Three interventions in animal models to rationally alter LDL composition lowered its susceptibility to aggregate and slowed atherosclerosis. Similar compositional changes induced in humans by PCSK9 inhibition or healthy diet also lowered LDL aggregation susceptibility. Aggregated LDL in vitro activated macrophages and T cells, two key cell types involved in plaque progression and rupture. Conclusion: Our results identify the susceptibility of LDL to aggregate as a novel measurable and modifiable factor in the progression of human ASCVD