A loss-of-function polymorphism in the propeptide of lysyl oxidase exacerbates atherosclerosis
In-Hyuk Jung, Junedh M. Amrute, Sofia E. Luna, Ryan E. Wagoner, Arturo Alisio, Paul C. Lee, Kendall H. Burks, Joohee Oh, Hannah C. Plunkett Paletta, Chul Joo Kang, Nathan O. Stitziel
In-Hyuk Jung, Junedh M. Amrute, Sofia E. Luna, Ryan E. Wagoner, Arturo Alisio, Paul C. Lee, Kendall H. Burks, Joohee Oh, Hannah C. Plunkett Paletta, Chul Joo Kang, Nathan O. Stitziel
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Research Article Cardiology Vascular biology

A loss-of-function polymorphism in the propeptide of lysyl oxidase exacerbates atherosclerosis

Abstract

A single-nucleotide missense polymorphism (rs1800449, R158Q) in the propeptide domain of lysyl oxidase (LOX-PP) is associated with increased risk of coronary artery disease (CAD) independent of changes in plasma lipid levels. Although the enzymatic function of LOX has an essential role for the cross-linking of extracellular matrix proteins in connective tissues, whether and how LOX-PP R158Q contributes to the development of atherosclerosis has not been clearly established. Here, hypercholesterolemia was induced in mice that were WT or homozygous for the LOX-PP R158Q polymorphism by adeno-associated virus-8–mediated overexpression of Pcsk9 followed by high-fat diet feeding for 16 weeks. We found that the R158Q polymorphism promoted atherosclerosis and induced proliferation of macrophages and vascular smooth muscle cells without altering LOX enzymatic activity. Using single-cell RNA sequencing, we found the transcriptional program of atherosclerotic plaques from mice harboring R158Q was strongly enriched for proliferation- and calcification-related genes in a regionally distinct manner. Together, these results establish an enzymatically independent proatherogenic role for the LOX-PP and suggest its potential as a novel therapeutic target.

Authors

In-Hyuk Jung, Junedh M. Amrute, Sofia E. Luna, Ryan E. Wagoner, Arturo Alisio, Paul C. Lee, Kendall H. Burks, Joohee Oh, Hannah C. Plunkett Paletta, Chul Joo Kang, Nathan O. Stitziel

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Figure 1

R158Q polymorphism in LOX-PP promotes atherosclerosis and proliferation of VSMCs and macrophages.

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R158Q polymorphism in LOX-PP promotes atherosclerosis and proliferation ...
(A) Body weight of LoxWT and LoxR152Q male mice during HFD feeding (n = 9–12/group). (B) Plasma total cholesterol at 2, 8, and 16 weeks of HFD feeding (n = 9–12/group), (C) triglyceride (n = 9–10/group), and (D) glucose (n = 9–12/group) after 16 weeks of HFD feeding. (E) En face Oil Red O–stained aortas. (F) Quantification of Oil Red O–stained area in each aortic arch and whole aorta (n = 9–12/group). (G) Oil Red O–stained aortic root cross sections. (H) Quantification of Oil Red O–stained atherosclerotic plaque area from 8 sections taken at 5, 20, 35, 100, 115, 130, 195, 210 μm (section number 1 through 8) from the aortic root toward the top (n = 9–11/group). (I) Average plaque area was calculated from the values measured across the 8 sections in H. (J) Necrotic core of aortic roots outlined by H&E staining. Red arrows indicate necrotic core in plaque from each genotype. (K) Quantification of necrotic core as a percentage of plaque area (n = 7–10/group). (L) EdU staining in VSMCs and macrophages of the aortic root from LoxWT and LoxR152Q mice after 16 weeks of HFD feeding. Tissues were costained for SMα-actin and Mac3. Outlined areas indicate the regions magnified in the next panels. Yellow circles indicate EdU+ cells. (M) Quantification of EdU+ in SMα-actin+, Mac3+, and all DAPI+ cells (n = 6–7/group). Scale bars: 500 μm (G), 200 μm (J), and 50 μm (L). Data were analyzed with 2-way ANOVA test (A, B, and H) or unpaired nonparametric Mann-Whitney U test (C, D, F, I, K, and M) and are shown as the mean ± SEM. P, plaque; L, lumen; M, media.