Inhibition of the Ubiquitin-Activating Enzyme UBA1 Suppresses Diet-Induced Atherosclerosis in Apolipoprotein E-Knockout Mice

Abstract

Background: Ubiquitin-like modifier activating enzyme 1 (UBA1) is the first and major E1 activating enzyme in ubiquitin activation, the initial step of the ubiquitin-proteasome system. Defects in the expression or activity of UBA1 correlate with several neurodegenerative and cardiovascular disorders. However, whether UBA1 contributes to atherosclerosis is not defined.

Methods and results: Atherosclerosis was induced in apolipoprotein E-knockout (Apoe-/-) mice fed on an atherogenic diet. UBA1 expression, detected by immunohistochemical staining, was found to be significantly increased in the atherosclerotic plaques, which confirmed to be mainly derived from lesional CD68⁺ macrophages via immunofluorescence costaining. Inactivation of UBA1 by the specific inhibitor PYR-41 did not alter the main metabolic parameters during atherogenic diet feeding but suppressed atherosclerosis development with less macrophage infiltration and plaque necrosis. PYR-41 did not alter circulating immune cells determined by flow cytometry but significantly reduced aortic mRNA levels of cytokines related to monocyte recruitment (Mcp-1, Vcam-1, and Icam-1) and macrophage proinflammatory responses (Il-1β and Il-6). Besides, PYR-41 also suppressed aortic mRNA expression of NADPH oxidase (Nox1, Nox2, and Nox4) and lesional oxidative stress levels, determined by DHE staining. In vitro, PYR-41 blunted ox-LDL-induced lipid deposition and expression of proinflammatory cytokines (Il-1β and Il-6) and NADPH oxidases (Nox1, Nox2, and Nox4) in cultured RAW264.7 macrophages.

Conclusions: We demonstrated that UBA1 expression was upregulated and mainly derived from macrophages in the atherosclerotic plaques and inactivation of UBA1 by PYR-41 suppressed atherosclerosis development probably through inhibiting macrophage proinflammatory response and oxidative stress. Our data suggested that UBA1 might be explored as a potential pharmaceutical target against atherosclerosis.

Figure 1

UBA1 expression was upregulated and mainly derived from macrophages in the atherosclerotic plaques of Apoe-/- mice. (a) Expression of UBA1 in the vessels with (right) or without atherosclerotic plaques (left). (b) Colocalization of UBA1 with the macrophage marker CD68 in the atherosclerotic plaques.

Figure 2

Inhibition of UBA1 did not alter the main metabolic parameters during diet-induced atherogenesis in Apoe-/- mice. (a) Plasma total cholesterol and triglyceride levels before and after the atherogenic diet feeding. (b) Plasma lipoprotein profiles fractioned by FPLC before (left) and after (right) the atherogenic diet feeding. (c) Plasma glucose levels before and after the atherogenic diet feeding. (d) Insulin resistance detected by the glucose tolerance test after 8 weeks on the atherogenic diet. (e) Body weight gain during the atherogenic diet feeding. n = 6 per group; ns: not significant; ATD: atherogenic diet.

Figure 3

Inhibition of UBA1 attenuated diet-induced atherosclerosis in Apoe-/- mice. (a, b) Oil red O staining and quantitation of the atherosclerotic burden in the en face aorta (a) and the aortic root (b). (c) CD68 immunochemical staining and quantitation of the lesional macrophages in the aortic root. (d) α-SMA immunochemical staining and quantitation of the lesional smooth muscle cells in the aortic root. (e) HE staining and quantitation of the lesional necrotic areas in the aortic root. n = 6 per group; ∗: <0.05; ns: not significant.

Figure 4

Inhibition of UBA1 decreased proinflammatory cytokine levels in diet-induced atherosclerosis in Apoe-/- mice. (a) Flow cytometry analysis of circulating monocytes, neutrophils, and T cells. (b) Quantitative real-time PCR analysis of aortic expression of cytokines associated with monocyte recruitment (Mcp-1, Vcam-1, and Icam-1). (c) Quantitative real-time PCR analysis of aortic expression of macrophage-derived proinflammatory (Il-1β, Il-6, and Tnfα) and anti-inflammatory (Il-4 and Il-10) cytokines. n = 5‐6 per group; ∗: <0.05; ns: not significant.

Figure 5

Inhibition of UBA1 decreased cellular oxidative stress in diet-induced atherosclerosis in Apoe-/- mice. (a) Quantitative real-time PCR analysis of aortic expression of NADPH oxidases (Nox1, Nox2, and Nox4). (b) DHE staining and quantitation of the lesional oxidative stress level in the aortic root. n = 5 per group; ∗: <0.05.

Figure 6

Inhibition of UBA1 blunted ox-LDL-induced lipid accumulation and expression of proinflammatory cytokines and NADPH oxidases in cultured macrophages. (a) Oil red O staining of ox-LDL-treated RAW264.7 cells. (b) Quantitative real-time PCR analysis of proinflammatory (Il-1β, Il-6, and Tnfα) and anti-inflammatory (Il-4 and Il-10) cytokine expression in ox-LDL-treated RAW264.7 cells. (c) Quantitative real-time PCR analysis of NADPH oxidase (Nox1, Nox2, and Nox4) expression in ox-LDL-treated RAW264.7 cells. n = 3 per group; ∗: <0.05; ∗∗: <0.01; ns: not significant.