Augmented EGF receptor tyrosine kinase activity impairs vascular function by NADPH oxidase-dependent mechanism in type 2 diabetic mouse

Abstract

We previously determined that augmented EGFR tyrosine kinase (EGFRtk) impairs vascular function in type 2 diabetic mouse (TD2). Here we determined that EGFRtk causes vascular dysfunction through NADPH oxidase activity in TD2. Mesenteric resistance arteries (MRA) from C57/BL6 and db−/db− mice were mounted in a wired myograph and pre-incubated for one hour with either EGFRtk inhibitor (AG1478) or exogenous EGF. The inhibition of EGFRtk did not affect the contractile response to phenylephrine-(PE) and thromboxane-(U46619) or endothelium-dependent relaxation (EDR) to acetylcholine in MRA from control group. However, in TD2 mice, AG1478 reduced the contractile response to U46619, improved vasodilatation and reduced p22phox-NADPH expression, but had no effect on the contractile response to PE. The incubation of MRA with exogenous EGF potentiated the contractile response to PE in MRA from control and diabetic mice. However, EGF impaired the EDR and potentiated the vasoconstriction to U46619 only in the control group. Interestingly, NADPH oxidase inhibition in the presence of EGF restored the normal contraction to PE and improved the EDR but had no effect on the potentiated contraction to U46619. Vascular function improvement was associated with the rescue of eNOS and Akt and reduction in phosphorylated-RhoKinase, NOX4 mRNA levels and NADPH oxidase activity. MRA from p47phox−/− mice incubated with EGF potentiated the contraction to U46619 but no effect to PE or ACh responses. The present study provides evidence that augmented EGFRtk impairs vascular function by NADPH oxidase-dependent mechanism. Therefore, EGFRtk and oxidative stress should be potential targets to treat vascular dysfunction in TD2.

Microvascular NADPH oxidase in health and disease

“…For example, mRNA transcript and protein of Nox1, Nox2, Nox4, p22 phox , p47 phox , p67 phox and Rac1/2 subunits were detected in mouse and rat mesenteric resistance arteries [19,58,60,66,67,71], where Noxs are implicated in structural and functional alterations in response to distinct stimuli [17,72,73]. In mouse coronary arteries, protein expression of Nox2 and its regulatory subunits p67 phox , p47 phox and p22 phox were reported [64].…”

“…The product of this reaction, peroxynitrite, is in and of itself a powerful oxidant and can exacerbate vascular dysfunction by causing further damage to lipids, proteins and DNA, uncoupling endothelial nitric oxide synthase (eNOS) and diminishing smooth muscle responses to NO [111,112]. In the systemic microvasculature, impaired endothelium-dependent vasodilation and concomitant enhanced Nox-mediated oxidative stress have been observed in microvascular coronary arterioles and mesenteric resistance arteries in a number of cardiovascular diseases, such as obesity [113,114], diabetes [18,19,47,115,116], hypertension [117–119] and ischemia-reperfusion [73]. In these studies, elevated expression of Nox subunits is a common characteristic across different cardiovascular pathologies.…”

“…ROS production is even more pronounced in aged diabetic rats, in which elevation in p67 phox subunit expression was also noted [47]. In other type 2 diabetic mouse models, upregulation of other Nox2 subunits, namely p22 phox , p40 phox and p47 phox , is observed in mesenteric and coronary resistance arteries [18,19,115]. Further corroborating a role of Nox2 in endothelial dysfunction, gp91ds-tat (aka Nox2ds-tat)-treated arterioles exhibited improved vasodilation to acetylcholine compared to untreated vessels.…”

“…As a primary source for oxidative stress and reactive oxygen species (ROS) in vascular cells, the contribution of NADPH oxidases (Noxs) to the cardiovascular system in health and disease has been investigated in a plethora of studies [9–11,13–15]. In the microcirculation, Noxs are implicated in a variety of vascular pathologies including arteriolar remodeling [16,17] and endothelial dysfunction [18,19] in systemic microvessels, as well as impaired neurovascular coupling [20] and disrupted blood-brain barrier (BBB) integrity [21] in the cerebral circulation. The current review aims not to be an exhaustive review on the roles of all sources of oxidases in the microcirculation.…”

“…Being the first to be discovered, Nox2 remains to be the most extensively studied member of the Nox family in numerous physiological and pathological processes. In the microvasculature, Nox2 appears to be mostly present in the endothelium and adventitia [20,24], responsible for endothelial dysfunction under disease conditions [19,47] but has been shown by Touyz et al to also be functionally active in human resistance smooth muscle cells [43]. Similar to Nox2, the canonical Nox1 requires binding of p22 phox , p47 phox homologue NoxO1, p67 phox homologue NoxA1 and Rac1/2 for activation [48].…”