Lysyl oxidase-like 2 depletion is protective in age-associated vascular stiffening

Abstract

Vascular stiffening and its sequelae are major causes of morbidity and mortality in the elderly. The increasingly accepted concept of "smooth muscle cell (SMC) stiffness syndrome" along with matrix deposition has emerged in vascular biology to account for the mechanical phenotype of arterial aging, but the molecular targets remain elusive. In this study, using an unbiased proteomic analysis, we identified lysyl oxidase-like 2 (LOXL2) as a critical SMC mediator for age-associated vascular stiffening. We tested the hypothesis that loss of LOXL2 function is protective in aging-associated vascular stiffening. We determined that exogenous and endogenous nitric oxide markedly decreased LOXL2 abundance and activity in the extracellular matrix of isolated SMCs and LOXL2 endothelial cells suppress LOXL2 abundance in the aorta. In a longitudinal study, LOXL2^+/- mice were protected from age-associated increase in pulse-wave velocity, an index of vascular stiffening, as occurred in littermate wild-type mice. Using isolated aortic segments, we found that LOXL2 mediates vascular stiffening in aging by promoting SMC stiffness, augmented SMC contractility, and vascular matrix deposition. Together, these studies establish LOXL2 as a nodal point for a new therapeutic approach to treat age-associated vascular stiffening. NEW & NOTEWORTHY Increased central vascular stiffness augments risk of major adverse cardiovascular events. Despite significant advances in understanding the genetic and molecular underpinnings of vascular stiffening, targeted therapy has remained elusive. Here, we show that lysyl oxidase-like 2 (LOXL2) drives vascular stiffening during aging by promoting matrix remodeling and vascular smooth muscle cell stiffening. Reduced LOXL2 expression protects mice from age-associated vascular stiffening and delays the onset of isolated systolic hypertension, a major consequence of stiffening.

Keywords: aging; lysyl oxidase-like 2; pulse-wave velocity; vascular stiffness.

Fig. 1.

Analysis of the human aortic smooth muscle cell (HASMC) secretome. A: HASMCs were cocultured with or without human aortic endothelial cells (HAECs) in cell-culture inserts. The conditioned cell culture medium from the HASMC layer was collected and used for iTRAQ analysis. B: proteins secreted by HASMCs were classified based on function. C: volcano plot showing proteins with 2-fold or greater increase or decrease in secretion by the HASMC layer with P < 0.05 vs. control.

Fig. 2.

Endogenous and exogenous nitric oxide (NO) regulate extracellular lysyl oxidase-like 2 (LOXL2) abundance and activity in vascular smooth muscle cells (VSMCs). Ai–Aiv: representative Western blot (Ai), densitometry analysis of LOXL2 protein in the cell-derived extracellular matrix (ECM; Aii), conditioned cell culture media (CCM; Aiii), and LOXL2 activity in the CCM (Aiv) in the human aortic smooth muscle cells (HASMCs) in the presence or absence of human aortic endothelial cell (HAEC) coculture and the endothelial nitric oxide synthase inhibitor nitro-l-arginine methyl ester (l-NAME; 100 μM; n = 6, *P < 0.05, **P < 0.01 by Kruskal Wallis test with Dunn’s post hoc test). IB, immunoblot. Bi and Bii: representative Western blots of LOXL2 protein in the aortic matrix of intact and endothelium-denuded young (3–4 mo old) and old (18+ mo old) wild-type (WT) mice (Bi; n = 5 in each group; *P < 0.05, **P < 0.01, and ***P < 0.001 by 2-way ANOVA); basal NO production was lower in the aorta of old WT mice when compared with young (Bii; n = 5 in each group, *P < 0.05, **P < 0.01 by Wilcoxon rank sum test). Ci–Civ: representative Western blot (Ci) of LOXL2 protein abundance and densitometry analysis in the ECM (Cii), CCM (Ciii), and LOX activity in the CCM (Civ) of HASMCs with increasing concentration of the nitric oxide donor S-nitrosoglutathione (GSNO; n = 6, *P < 0.05, **P < 0.01 by Kruskal Wallis test with Dunn’s post hoc test). RFU, relative fluorescence units.

Fig. 3.

Expression of lysyl oxidase (LOX) proteins in LOXL2^+/− mice. Representative Western blots comparing the expression of LOX, LOXL2, and LOXL3 in the heart, lung, uterus, aorta, and liver of wild-type (WT) and littermate LOXL2^+/− mice with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was as a loading control. (n = 6 mice). ND, not detected.

Fig. 4.

Lysyl oxidase-like 2 (LOXL2) knockdown is protective against age-associated vascular stiffening in mice. A: pulse-wave velocity (PWV) with increasing age in the LOXL2^+/− mice (n = 12; 7 female, 5 male) and littermate wild-type (WT) mice (n = 10; 5 female, 5 male; n = 20–35 measures per age for each cohort; *P < 0.05, **P < 0.01 vs. WT 1–4 mo old; ‡P < 0.05 vs. WT 4–8 mo old; #P < 0.05, ##P < 0.01 vs. age-matched LOXL2^+/− littermates by 2-way ANOVA). B: systolic pressure with increasing age in WT (n = 10; 5 female, 5 male) and LOXL2^+/− littermate mice (n = 12; 7 female, 5 male) (n = 25–35 measurements per age group per genotype; **P < 0.01 vs WT 1–4 mo old; ###P < 0.001 vs. age-matched LOXL2 littermates by 2-way ANOVA). C: representative Western blots for LOXL2 protein expression in the aortic matrix of young (Y) and old (O) LOXL2^+/− mice and WT littermates (images are representative of n = 5 in each group; old = 20–22 mo old, young = 3–4 mo old; *P < 0.05 vs. old WT by 2-way ANOVA). ECM, extracellular matrix. D: representative confocal microscopy images of intact aortas from 15+-mo-old WT and LOXL2^+/− mice (n = 5) stained for LOXL2 (red) and nuclei (blue). Scale bars = 50 μm.

Fig. 5.

Immunohistochemical analysis of young (3–4 mo old) and old (22 mo old) wild-type (WT) and lysyl oxidase-like 2 (LOXL2) littermate mouse aorta. All images shown are representative of images from 6 independent mouse aortas (×10 magnification) as whole rings. Insets: ×40 for each specimen. A–V: hematoxylin and eosin (H&E) stain (A), Masson trichrome stain (B), and Movat pentachrome stain (C). Scale bars = 50 μm. D: aging-related changes in nuclear content and wall thickness of aortas of WT and LOXL2^+/− mice. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 6.

Lysyl oxidase-like 2 (LOXL2) promotes aortic matrix stiffness and vascular smooth muscle cell (VSMC) stiffness in aging. A: passive compliance of the carotid artery, measured by pressure myography, aged [old (O)] wild-type (WT; 15 mo old, n = 7; 3 female, 4 male), young (Y) WT (3–4 mo old, n = 10; 5 female, 5 male), young LOXL2^+/− (3-4 mo old, n = 12; 7 female, 5 male) and aged LOXL2^+/− (15 mo old, n = 12; 7 female, 5 male) mice (*P < 0.05 by Kruskal-Wallis test with Dunn’s correction). Bi and Bii: tensile testing of intact (Bi) and decellularized (Bii) aortic segments of WT and littermate LOXL2^+/− mice. Data are shown as means (solid line) ± SE (broken lines) (n = 12 LOXL2^+/− and n= 8 WT mice; ***P < 0.0001 by Wilcoxon rank sum test). C: LOXL2 expression in the conditioned media and cell-derived ECM of endothelial and smooth muscle cells isolated from LOXL2^+/− and littermate WT mouse aorta; GAPDH is shown as a reference control. Blot is representative of 5 independent experiments. IB, immunoblot. D: stiffness of mouse aortic smooth muscle cells isolated from old (15 mo old) WT and LOXL2^+/− littermate mice measured by magnetic torsion cytometry (n = 543 WT cells; 165 LOXL2^+/− cells; *P < 0.05 by Student’s t-test). E: cytoskeletal remodeling dynamics of aortic smooth muscle cells from old (15 mo old) WT and littermate LOXL2^+/− mice (n = 599 WT cells, 586 LOXL2^+/− cells; ****P < 0.05 by Student’s t-test). MSD, mean square displacement. F: motility of WT- and LOXL2-depleted VSMC examined by transwell migration assay using 10% serum as chemoattractant (n = 6; **P < 0.01 by 2-way ANOVA). RFU, relative fluorescence units. G: deadhesion dynamics of WT- and LOXL2-depleted VSMCs using 0.05% trypsin (n = 5; ****P < 0.0001 by Wilcoxon rank sum test).

Fig. 7.

Lysyl oxidase-like 2 (LOXL2) depletion decreases vascular smooth muscle cell (VSMC) contractility. A: representative phase contrast and traction map images of VSMCs from old (15 mo old) wild-type (WT) and LOXL2^+/− cells as measured by Fourier transform traction microscopy. The white lines show the cell boundary, and the colors show the magnitude of the traction force in Pascals indexed to the color bar at the right. Bi–Biv: computed projected area (Bi), traction stress (Bii), and derived strain energy (Biii), and prestress (Biv) of isolated VSMCs. For these studies, cells were plated onto an inert elastic gel (8 kPa) coated with type I collagen (WT, n = 18; LOXL2^+/−, n = 19 individual cell measurements). For data that were not normally distributed [root mean squares (RMS) traction, total strain energy, and prestress], statistical analyses were performed on transformed data (*P < 0.05, **P < 0.01 by Student’s t-test). C: constriction response of LOXL2^+/− and WT mouse aortic rings to increasing concentrations of phenylephrine (n = 8 per group; *P < 0.05 vs. WT at same phenylephrine concentration by Kruskal-Wallis test with Dunn’s post hoc correction). D and E: endothelial-independent relaxation of vessels pre-constricted with phenylephrine with increasing concentrations of sodium nitroprusside (n = 8; D) and endothelial-dependent relaxation of vessels preconstricted with phenylephrine with increasing concentrations of acetylcholine (n = 8; E) in LOXL2^+/− and WT mouse aortic rings.

Fig. 8.

Lysyl oxidase-like 2 (LOXL2) promotes vascular stiffening in aging. Aging results in endothelial dysfunction that in turn results in increased LOXL2 and soluble collagen in the vascular matrix. This leads to both increased matrix cross linking and vascular smooth muscle cell (VSMC) stiffness/tone. Together, these changes result in increased vascular stiffness. LOXL2 knockdown limits this process and prevents vascular stiffening with age, thus interrupting the onset of isolated systolic hypertension. NO, nitric oxide.