Increased soluble urokinase plasminogen activator levels modulate monocyte function to promote atherosclerosis

Abstract

People with kidney disease are disproportionately affected by atherosclerosis for unclear reasons. Soluble urokinase plasminogen activator receptor (suPAR) is an immune-derived mediator of kidney disease, levels of which are strongly associated with cardiovascular outcomes. We assessed suPAR's pathogenic involvement in atherosclerosis using epidemiologic, genetic, and experimental approaches. We found serum suPAR levels to be predictive of coronary artery calcification and cardiovascular events in 5,406 participants without known coronary disease. In a genome-wide association meta-analysis including over 25,000 individuals, we identified a missense variant in the plasminogen activator, urokinase receptor (PLAUR) gene (rs4760), confirmed experimentally to lead to higher suPAR levels. Mendelian randomization analysis in the UK Biobank using rs4760 indicated a causal association between genetically predicted suPAR levels and atherosclerotic phenotypes. In an experimental model of atherosclerosis, proprotein convertase subtilisin/kexin-9 (Pcsk9) transfection in mice overexpressing suPAR (suPARTg) led to substantially increased atherosclerotic plaques with necrotic cores and macrophage infiltration compared with those in WT mice, despite similar cholesterol levels. Prior to induction of atherosclerosis, aortas of suPARTg mice excreted higher levels of CCL2 and had higher monocyte counts compared with WT aortas. Aortic and circulating suPARTg monocytes exhibited a proinflammatory profile and enhanced chemotaxis. These findings characterize suPAR as a pathogenic factor for atherosclerosis acting at least partially through modulation of monocyte function.

Keywords: Atherosclerosis; Cardiology; Innate immunity.

Figure 1. Median CAC score at baseline and follow-up by suPAR categories.

Median CAC score (AU) based on Agatston scoring method at baseline and initial follow-up visits stratified by suPAR categories: 0–2.0 ng/mL, 2.0–2.5 ng/mL, 2.5–3.0 ng/mL, and >3.0 ng/mL. Error bars represent 95% CI.

Figure 2. Cumulative incidence of any CVD event by suPAR categories.

Unadjusted Kaplan-Meier curves for the cumulative incidence of CVD events stratified by suPAR categories: 0–2.0 ng/mL (red), 2.0–2.5 ng/mL (green), 2.5–3.0 ng/mL (blue), >3 ng/mL (purple). The difference in cumulative incidence curves between suPAR categories was tested using the log-rank test. A CVD event was defined as the composite of myocardial infarction, resuscitated cardiac arrest, angina, revascularization, stroke (excluding transient ischemic attack), and death due to CVD.

Figure 3. In vitro and in vivo expression of PLAUR missense variants and suPAR levels.

Human suPAR levels in (A) supernatant of HEK cells 48 hours after transfection with rs4760 (n = 3) and rs2302524 (n = 3) PLAUR variants and in (B) C57BL/6J mice 24 hours after hydrodynamic tail-vein injection of plasmid DNA containing WT (n = 6) or the rs2302524 (n = 10) or rs4760 (n = 7) variant. ***P < 0.001; ***P < 0.0001, 1-way ANOVA.

Figure 4. MR phenome-wide association of genetically predicted suPAR by rs4760 with CVD and rare damaging missense variants’ impact on the odds of ischemic heart disease.

(A) Causal effect of suPAR on 13 CVDs by MR using missense variant rs4760 as instrument. Effect estimates are provided per 1 SD increase in suPAR levels. P values were adjusted using the false discovery rate method. (B) Rare variant gene collapsing analysis of the more than 280,000 exomes in the UK Biobank. Both rare protein truncating variants and rare damaging missense variants in the PLAUR gene were selected to study the impact of attenuated PLAUR function on coronary heart disease.

Figure 5. suPAR overexpression leads to increased atherosclerotic and necrotic plaques in a murine model of atherosclerosis.

WT (n = 18) and suPAR^Tg (n = 21) mice were maintained on a low-fat diet until 3 months of age and were then transfected with Pcsk9-AAV and fed a western diet (WD) for 10 weeks. At this point, aortic roots were obtained, paraffin embedded, and stained with H&E and Mac2 (galectin 3). (A) Cross sections of aortic roots from C57BL/6 WT and suPAR^Tg mice show total lesion area, outlined in dashed lines, and necrotic core area, outlined in dotted lines. Higher magnification shows the presence of necrotic core. Mac2 monoclonal antibody stain shown on aortic sinus cross sections from WT and suPAR^Tg mice. Scale bars: 100 μm; 50 μm. (B and C) Quantification of total lesion area and necrotic core area for all 30 sections. (D) Quantification of Mac2 staining as a percentage of total plaque area with necrotic area subtracted. Atherosclerotic plaque and necrotic core areas: n = 18 WT and n = 21 suPAR^Tg groups. Tissue sections are 6 μm each with 6 μm blank section between for a total of one 360 μm through the aortic sinus. Each data point represents a biological replicate for D. **P < 0.01; ****P < 0.0001, 2-way ANOVA (B and C); Student’s t test (D).

Figure 6. suPAR overexpression in mice leads to proatherosclerotic phenotype in circulating and aortic monocytes.

Aortas and blood were harvested from disease-free C57BL/6 WT and suPAR overexpressing mice (suPAR^Tg mice). (A) Aortas from WT (n = 11) and suPAR^Tg (n = 11) mice were excised, cleaned of fat, and cultured for 24 hours. At this point, the conditioned culture medium was isolated and CCL2 level was assessed by ELISA. (B) Aortas from WT (n = 6) and suPAR^Tg (n = 6) mice were isolated, cleaned of fat, digested, stained with fluorescently labeled antibodies, and analyzed by flow cytometry. Quantification of F4/80–Ly-6G–CD11b⁺ monocytes from WT and suPAR^Tg mice as a percentage of live CD11b⁺CD45⁺ cells and median fluorescent intensity (MFI) of CCR2 expression from WT on F4/80–Ly-6G–CD11b⁺ monocytes. (C) Blood from WT and suPAR^Tg mice was isolated and red blood cells were lysed, stained with fluorescently labeled antibodies, and analyzed by flow cytometry. MFI on live CD45⁺CD11b⁺ monocytes for expression of CCR2, MHCII, and CX3CR1, and percentage of uPAR⁺ cells of live CD45⁺CD11b⁺ cells. CCR2: n = 16 WT and n = 15 suPAR^Tg, compared by Student’s t test. MHCII: n = 6 WT and n = 6 suPAR^Tg. CX3CR1: n = 4 WT and n = 4 suPAR^Tg. uPAR⁺ cells: n = 6 WT and n = 5 suPAR^Tg. For MHCII, CX3CR1, and uPAR⁺ cells, Mann-Whitney U test was used. (D) Monocytes were isolated from spleens of WT and suPAR^Tg mice and cultured in Transwell assays with either control cell culture media or cell culture media with CCL2 added. Quantification of fluorescent intensity of cellular dye was compared by 2-way ANOVA. n = 6 for each group. Each data point represents a biological replicate.