Upregulation of Nox4 by TGFβ1 Oxidizes SERCA and Inhibits NO in Arterial Smooth Muscle of the Prediabetic Zucker Rat

Abstract

Rationale: Vascular smooth muscle cell (SMC) migration is an important pathological process in several vascular occlusive diseases, including atherosclerosis and restenosis, both of which are accelerated by diabetes mellitus.Objective: To determine the mechanisms of abnormal vascular SMC migration in type 2 diabetes, the obese Zucker rat (ZO), a model of obesity and insulin resistance, was studied. Key Words: nitric oxide Ⅲ cell migration Ⅲ NADPH oxidase Ⅲ obese Zucker rat Ⅲ TGF-␤1 Ⅲ Smad2 D iabetic patients have a much higher morbidity and mortality from cardiovascular diseases, including atherosclerosis and restenosis, compared with other patients. Vascular smooth muscle cell (SMC) migration contributes significantly to these pathological processes. Generally, SMCs remain quiescent in the vasculature, but when the endothelial layer is disrupted, the underlying SMCs migrate from media to intima and form the neointima. This process is accelerated by diabetes mellitus. Numerous clinical studies have indicated that diabetic patients have a higher incidence of restenosis after percutaneous coronary interventions compared with patients without diabetes. [1][2][3] Nitric oxide (NO), the biologically active component of endothelium-derived relaxing factor, has critical roles in the maintenance of vascular homeostasis. The sarco-/ endoplasmic reticulum Ca 2ϩ ATPase (SERCA) plays a very important role in maintaining intracellular calcium levels by taking up calcium into sarco-/endoplasmic reticulum. Previous studies showed that NO decreases intracellular calcium, which causes SMC relaxation and inhibits growth and migration. Our previous studies showed that NO upregulates SERCA activity by S-glutathiolation of the most reactive thiol on cysteine-674 (C674) and thereby inhibits SMC migration. 4 Impaired NO-induced relaxation of atherosclerotic arteries or inhibition of migration of cultured SMCs exposed to high glucose was attributable to irreversible oxidation of SERCA C674, which prevents the S-glutathiolation and increase in SERCA activity required for the response to NO. 5 A recent report that vascular injury, which is normally inhibited by NO, is unaffected in Original Methods and Results:

Composition of the redox environment of the endoplasmic reticulum and sources of hydrogen peroxide

“…Nevertheless, association of Nox4 to the ER seems highly likely, but the exact function of the enzyme in this organelle remains to be clarified. The potential targets of Nox4-derived hydrogen peroxide in the ER include protein-tyrosine phosphatase 1B [117,118], sarco/endoplasmic reticulum Ca 2+ -ATPase [119] and ryanodine receptor [118].…”

Correction

“…1 We found that the adenoviral vector intended to express human wild type SERCA2b in the experiments shown in Figure 1b in this paper actually encoded the SERCA2b C674S mutant and in addition was contaminated with E1A adenovirus. The incorrect adenoviral vector was identified by PCR of the full-length SERCA present in the viral DNA within infected cells.…”

“…The methods are provided in full in Tong et al 1 and Haeussler et al 2 Titers of the new wild type SERCA 2b and the SERCA 2b C674S mutant were adjusted so that both increased mRNA levels 2-to 3-fold over baseline with an equal increase in SERCA protein expression confirmed by western blot. In the figure below, cultured Obese Zucker rat aorta smooth muscle cells infected with an "empty" adenoviral vector, which lacks a gene insert, migrated similarly in response to serum as the cells infected with the LacZ vector used as control in the original Figure 1b of Tong et al Cells infected with authentic wild type SERCA2b showed similar migration, but demonstrated a restoration of the ability of the nitric oxide donor, DETA NONOate, to inhibit their migration into a scratch wound (see Figure 1 below).…”

Vascular Nox4

“…9,10 For example, in obese Zucker rats, a model of obesity and insulin resistance, common carotid injury was attenuated by Nox4 knockdown with Nox4 short hairpin RNA, implicating Nox4 in neointimal formation and vascular injury. 10 Pharmacological inhibitors of Nox4 (VAS2870 and GKT136901) also prevented agonist-induced oxidative stress and inflammatory responses in cultured cells. 11,12 However, these observations are merely suggestive of Nox4 in vascular pathology and by no means establish cause and effect.…”

“…First, in the study of Schroder et al, 14 vascular functional studies were performed in large conduit vessels (aorta) and in lung endothelial cells from Nox4-deficient mice, whereas many previous studies explored small arteries and cultured vascular smooth muscle cells. 3,5,9,10,11,17,18 Second, in Nox4 knockout mice, the gene deletion is global, and, accordingly, it is not entirely correct to implicate a vascular-specific pathology in a model in which there is a ubiquitous knockout. Third, the fact that it is only in challenged conditions that Nox4 knockout mice exhibit a vascular phenotype indicates a nonessential role of Nox4 in physiological conditions.…”