NOX4-derived reactive oxygen species limit fibrosis and inhibit proliferation of vascular smooth muscle cells in diabetic atherosclerosis

Abstract

Smooth muscle cell (SMC) proliferation and fibrosis contribute to the development of advanced atherosclerotic lesions. Oxidative stress caused by increased production or unphysiological location of reactive oxygen species (ROS) is a known major pathomechanism. However, in atherosclerosis, in particular under hyperglycaemic/diabetic conditions, the hydrogen peroxide-producing NADPH oxidase type 4 (NOX4) is protective. Here we aim to elucidate the mechanisms underlying this paradoxical atheroprotection of vascular smooth muscle NOX4 under conditions of normoand hyperglycaemia both in vivo and ex vivo. Following 20-weeks of streptozotocin-induced diabetes, Apoe−/− mice showed a reduction in SM-alpha-actin and calponin gene expression with concomitant increases in platelet-derived growth factor (PDGF), osteopontin (OPN) and the extracellular matrix (ECM) protein fibronectin when compared to non-diabetic controls. Genetic deletion of Nox4 (Nox4−/− Apoe−/− ) exacerbated diabetes-induced expression of PDGF, OPN, collagen I, and proliferation marker Ki67. Aortic SMCs isolated from NOX4-deficient mice exhibited a dedifferentiated phenotype including loss of contractile gene expression, increased proliferation and ECM production as well as elevated levels of NOX1-associated ROS. Mechanistic studies revealed that elevated PDGF signalling in NOX4-deficient SMCs mediated the loss of calponin and increase in fibronectin, while the upregulation of NOX1 was associated with the increased expression of OPN and markers of proliferation. These findings demonstrate that NOX4 actively regulates SMC pathophysiological responses in diabetic Apoe−/− mice and in primary mouse SMCs through the activities of PDGF and NOX1.

Reactive Oxygen Species Generation and Atherosclerosis

“…27 Nox4 deletion further increased aortic expression of PDGF, collagen I, and Ki-67. SMCs isolated from Nox4-deficient animals had lower expression of smooth muscle contractile markers, produced less H 2 O 2 but more superoxide and upregulated Nox1.…”

“…Abnormal expression of PDGF-BB in Nox4 −/− SMCs could also be reversed by PDGF inhibition or treatment with H 2 O 2 . 27 What is more, Nox4 together with Nrf2 play significant roles in the CD38 signaling pathway, which is crucial for the maintenance of SMC contractile phenotype. 45 Coronary arterial myocytes isolated from Cd38 −/− mice had lower calponin and SM22α expression, accompanied by an increase in vimentin and PCNA (proliferating cell nuclear antigen).…”

“…25 On the other hand, Nox4 knockout aggravated atherosclerosis, 26 especially in diabetic mice. 27 High levels of oxidative stress can be counteracted by complex antioxidant cell systems that are crucial for the maintenance of redox balance. The key player that plays a primary role in the regulation of antioxidant gene response is nuclear factor erythroid 2-related factor 2 (Nrf2), 28 encoded by the Nfe2l2 gene.…”

Hydrogen peroxide formation by Nox4 limits malignant transformation

“…In previous work we reported that Nox4 maintains the expression of Nrf2 (9). Moreover, Nox4-/-mice exhibit a hyperinflammatory response in the vasculature which results in cardiovascular dysfunction and accelerated arteriosclerosis (9)(10)(11)(12)(13). We and others also found Nox4 to be induced in the course of differentiation and to be essentially involved in the differentiation process of mesenchymal cells (14)(15)(16).…”

NADPH oxidase 4 and its role in the cardiovascular system

“…Moreover, oxidized LDL stimulates NOX4 expression in macrophages, a process that leads to necrotic core formation within lesions (48). Furthermore, NOX4 has been linked to smooth muscle cell (SMC) migration and proliferation, which are essential steps in the development of atherosclerosis (42,49). Xu et al (43) reported that NOX4 expression was increased in aged atherosclerotic plaques, specifically in the SMC of unstable plaques, through an increase in SMC senescence and apoptosis (43), an important step in the development of unstable lesions.…”